Conditionally Active Biological Proteins

ABSTRACT

This disclosure relates to a method of generating conditionally active biologic proteins from wild type proteins, in particular therapeutic proteins, which are reversibly or irreversibly inactivated at some physiological conditions. For example, conditionally active biologic proteins are active in tumors, but virtually inactive at other body parts, or conditionally active antibodies capable of crossing blood-brain-barrier.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 15/308,659, filed on Nov. 3, 2016, which, in turn is a 371continuation of International application no. PCT/US2015/030086, filedMay 11, 2015, which, in turn, claims the benefit of U.S. Provisionalapplication No. 62/153,001, filed Apr. 27, 2015, U.S. Provisionalapplication No. 62/043,080, filed Aug. 28, 2014, and U.S. Provisionalapplication No. 61/992,415, filed May 13, 2014.

FIELD OF THE DISCLOSURE

This disclosure relates to the field of protein evolution and activity.Specifically, this disclosure relates to a method of generatingconditionally active biologic proteins from wild type proteins, inparticular therapeutic proteins, which are reversibly or irreversiblyinactivated at the wild type normal physiological conditions as well asto such conditionally active biologic proteins and uses of suchconditional active biologic proteins.

BACKGROUND OF THE DISCLOSURE

There is a considerable body of literature describing the potential forevolving proteins for a variety of characteristics, especially enzymesfor example, to be stabilized for operation at different conditions. Forexample, enzymes have been evolved to be stabilized at highertemperatures, with varying activity. In situations where there is anactivity improvement at the high temperature, a substantial portion ofthe improvement can be attributed to the higher kinetic activitycommonly described by the Q10 rule where it is estimated that in thecase of an enzyme the turnover doubles for every increase of 10 degreesCelsius. In addition, there exist examples of natural mutations thatdestabilize proteins at their normal operating conditions, such aswild-type temperature activity of the molecule. For temperature mutants,these mutants can be active at the lower temperature, but typically areactive at a reduced level compared to the wild type molecules (alsotypically described by a reduction in activity guided by the Q10 orsimilar rules).

It is desirable to generate useful molecules that are conditionallyactivated, for example virtually inactive at wild-type conditions butare active at other than wild-type conditions at a level that is equalor better than at wild-type conditions, or that are activated orinactivated in certain microenvironments, or that are activated orinactivated over time. Besides temperature, other conditions for whichthe proteins can be evolved or optimized include at least pH, osmoticpressure, osmolality, oxidation and electrolyte concentration. Otherdesirable properties that can be optimized during evolution includechemical resistance, and proteolytic resistance.

Many strategies for evolving or engineering molecules have beenpublished. US 2010/0189651 discloses an engineered antibody containingan antibody or antibody fragment linked with a masking moiety. Such anengineered antibody can be further coupled to a cleavable moiety,resulting in an antibody that can be conditionally activated. Thecleavable moiety is capable of being cleaved, reduced, or photolysed.The antibody can exhibit a conformation such that the antibody is moreaccessible to a target after removal of the masking moiety by cleavage,reduction, or photolysis of the cleavable moiety.

US 2013/0101555 discloses engineered activatable proproteincompositions. An activatable proprotein contains a functional proteincoupled to a peptide mask, and further coupled to an activatable linker.In a non-activated state, the peptide mask inhibits binding of thefunctional protein to its target or binding partner. In an activatedstate, the peptide mask does not inhibit binding of the functionalprotein to its target or binding partner. Proproteins can provide forreduced toxicity and adverse side effects that could otherwise resultfrom binding of a functional protein at non-treatment sites if it werenot inhibited from binding to its binding partner at such non-treatmentsites. Proproteins containing the peptide mask can also have a longer invivo or serum half-life than the corresponding functional protein notcontaining the peptide mask.

US 2011/0229489 discloses antibodies with pH dependent binding toantigens such that the affinity for antigen binding at physiological pH(i.e., pH 7.4) is greater than at endosomal pH (i.e., pH 6.0 or 5.5).Such pH-dependent antibodies preferentially dissociate from the antigenin the endosome. This can increase antibody half-life, as compared toantibodies with equivalent K_(DS) at pH 7.4 but with no pH dependentbinding, when the antigen is one that undergoes antigen-mediatedclearance (e.g., PCSK9). Antibodies with pH-dependent binding candecrease total antigen half-life when the antigen undergoes reducedclearance after being bound to an antibody.

US 2013/0266579 discloses a conditionally active anti-EGFR antibody. Theanti-EGFR antibody exhibits a ratio of binding activity to humanepidermal growth factor receptor (EGFR) for conditions in a tumorenvironment to conditions in a non-tumor environment of at least 3.0.The conditions in a tumor environment comprise one or both of a pH offrom 5.6 to 6.8 or a lactate concentration of from 5 mM to 20 mM, and aprotein concentration from 10 mg/mL to 50 mg/mL. The conditions in anon-tumor environment comprise one or both of a pH of from 7.0 to 7.8 ora lactate concentration of from 0.5 mM to 5 mM, and a proteinconcentration of from 10 mg/mL to 50 mg/mL. The anti-EGFR antibody issaid to be conditionally active under conditions that may be found in atumor microenvironment.

Pardoll et al, “The blockade of immune checkpoints in cancerimmunotherapy,” Nature Review Cancer, vol. 12, pages 252-264, 2012describes a cancer therapy that involves activating host anti-tumourimmunity by blockading host immune system checkpoints. Such a blockademay be achieved by inhibiting immune checkpoint proteins such asreceptors on T-cells, including cytotoxic T-lymphocyte-associatedantigen 4 (CTLA4) and death protein 1 (PD1). Antibodies against theseimmune checkpoint proteins have been developed for cancer therapy.

Engineering or evolving a protein to be inactive or virtually inactive(less than 10% activity and especially 1% activity) at its wild typeoperating condition, while maintaining activity equivalent or betterthan its wild type condition at new conditions, requires that thedestabilizing mutation(s) co-exist with activity increasing mutationsthat do not counter the destabilizing effect. It is expected thatdestabilization would reduce the protein's activity greater than theeffects predicted by standard rules such as Q10, therefore the abilityto evolve proteins that work efficiently at lower temperature, forexample, while being inactivated under their normal operating condition,creates an unexpected new class of conditionally active proteins.

Throughout this application, various publications are referenced byauthor and date. The disclosures of these publications in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art as known to thoseskilled therein as of the date of the disclosure described and claimedherein.

SUMMARY OF THE DISCLOSURE

In one aspect, the present invention provides a method of preparing aconditionally active biological protein, the method comprising the stepsof: i. selecting wild-type biological protein; ii. evolving the DNAwhich encodes the wild-type biological protein using one or moreevolutionary techniques to create mutant DNAs; iii. expressing themutant DNAs to obtain mutant biological proteins; iv. subjecting themutant biological proteins and the wild-type biological protein to anassay under a first physiological condition selected from physiologicalconditions of a first location selected from the group consisting ofsynovial fluid, a tumor microenvironment and a stem cell niche, and toan assay under a second physiological condition selected fromphysiological conditions of a second location in a body that isdifferent from the first location; and v. selecting the conditionallyactive biologic protein from the mutant biologic proteins which exhibitboth (a) an increased activity in the assay under the firstphysiological condition compared to the wild-type biologic protein, and(b) a decreased activity in the assay under the second physiologicalcondition compared to the wild-type biologic protein.

In another aspect, the present invention provides a method of preparinga conditionally active antibody for crossing the blood-brain barrier,the method comprising the steps of: i. selecting a wild-type antibodyagainst a blood-brain barrier receptor; ii. evolving the DNA whichencodes the wild-type antibody using one or more evolutionary techniquesto create mutant DNAs; iii. expressing the mutant DNAs to obtain mutantantibodies; iv. subjecting the mutant antibodies and the wild-typeantibody to an assay under a first physiological condition in bloodplasma and to an assay under a second physiological condition in brainextracellular fluid; and v. selecting the conditionally active antibodyfrom the mutant antibodies which exhibit both: (a) a decrease inaffinity to the blood-brain barrier receptor in the assay under thefirst physiological condition compared to the wild-type antibody, and(b) an affinity selected from the group consisting of an increasedaffinity to the blood-brain barrier receptor in the assay under thesecond physiological condition and no affinity to the blood-brainbarrier receptor in the assay under the second physiological condition.

In yet another aspect, the method of the present invention furthercomprises the step of conjugating the conditionally active biologicalprotein to a molecule.

In yet another aspect, the method of the present invention furthercomprises the step of introducing at least one amino acid substitutionin the Fc region of a conditionally active antibody.

In yet another aspect, the method of the present invention furthercomprises the step of engineering the conditionally active antibody tobe multispecific.

In yet another aspect, the present invention provides a conditionallyactive biological protein. In yet another aspect, the conditionallyactive biological protein is a conditionally active antibody.

Definitions

In order to facilitate understanding of the examples provided herein,certain frequently occurring methods and/or terms will be definedherein.

As used herein in connection with a measured quantity, the term “about”refers to the normal variation in that measured quantity that would beexpected by the skilled artisan making the measurement and exercising alevel of care commensurate with the objective of the measurement and theprecision of the measuring equipment used. Unless otherwise indicated,“about” refers to a variation of +/−10% of the value provided.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, an array of spatially localized compounds (e.g.,a VLSIPS peptide array, polynucleotide array, and/or combinatorial smallmolecule array), biological macromolecule, a bacteriophage peptidedisplay library, a bacteriophage antibody (e.g., scFv) display library,a polysome peptide display library, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal (particularmammalian) cells or tissues. Agents are evaluated for potential enzymeactivity by inclusion in screening assays described herein below. Agentsare evaluated for potential activity as conditionally active biologictherapeutic enzymes by inclusion in screening assays described hereinbelow.

The term “amino acid” as used herein refers to any organic compound thatcontains an amino group (—NH₂) and a carboxyl group (—COOH); preferablyeither as free groups or alternatively after condensation as part ofpeptide bonds. The “twenty naturally encoded polypeptide-formingalpha-amino acids” are understood in the art and refer to: alanine (alaor A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp orD), cysteine (cys or C), gluatamic acid (glu or E), glutamine (gin orQ), glycine (gly or G), histidine (his or H), isoleucine (ile or I),leucine (leu or L), lysine (lys or K), methionine (met or M),phenylalanine (phe or F), proline (pro or P), serine (ser or S),threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), andvaline (val or V).

The term “amplification” as used herein means that the number of copiesof a polynucleotide is increased.

The term “antibody” as used herein refers to intact immunoglobulinmolecules, as well as fragments of immunoglobulin molecules, such asFab, Fab′, (Fab′)2, Fv, and SCA fragments, that are capable of bindingto an epitope of an antigen. These antibody fragments, which retain someability to selectively bind to an antigen (e.g., a polypeptide antigen)of the antibody from which they are derived, can be made using wellknown methods in the art (see, e.g., Harlow and Lane, supra), and aredescribed further, as follows. Antibodies can be used to isolatepreparative quantities of the antigen by immunoaffinity chromatography.Various other uses of such antibodies are to diagnose and/or stagedisease (e.g., neoplasia) and for therapeutic application to treatdisease, such as for example: neoplasia, autoimmune disease, AIDS,cardiovascular disease, infections, and the like. Chimeric, human-like,humanized or fully human antibodies are particularly useful foradministration to human patients.

An Fab fragment consists of a monovalent antigen-binding fragment of anantibody molecule, and can be produced by digestion of a whole antibodymolecule with the enzyme papain, to yield a fragment consisting of anintact light chain and a portion of a heavy chain.

An Fab′ fragment of an antibody molecule can be obtained by treating awhole antibody molecule with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain. Two Fab′ fragments are obtained per antibody molecule treated inthis manner.

An (Fab′)2 fragment of an antibody can be obtained by treating a wholeantibody molecule with the enzyme pepsin, without subsequent reduction.A (Fab′)2 fragment is a dimer of two Fab′ fragments, held together bytwo disulfide bonds.

An Fv fragment is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC”refers to a form of cytotoxicity in which secreted immunoglobulin bindsto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NaturalKiller (NK) cells, neutrophils, and macrophages) that enables thesecytotoxic effector cells to bind specifically to an antigen-bearingtarget cell and subsequently kill the target cell with cytotoxins.Ligand specific high-affinity IgG antibodies directed to the surface oftarget cells stimulate the cytotoxic cells via affinity to the ADCCdomain on the IgG to attack the cell bound to the IgG antibody via theFab region. Lysis of the target cell is extracellular, which requiresdirect cell-to-cell contact, and does not involve complement.

The ability of any particular antibody to mediate lysis of the targetcell by ADCC can be assayed. To assess ADCC activity, an antibody ofinterest is mixed with the target cells displaying the target ligand incombination with immune effector cells, which may be activated by theantigen antibody complexes resulting in cytolysis of the target cell.Cytolysis is often detected by the release of a label (e.g., radioactivesubstrates, fluorescent dyes or natural intracellular proteins) from thelysed cells. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells. Specificexamples of in vitro ADCC assays are described in Bruggemann et al,1987, J. Exp. Med., vol. 166, page 1351 ; Wilkinson et al, 2001, J.Immunol. Methods, vol. 258, page 183; Patel et al, 1995 J. Immunol.Methods, vol. 184, page 29 (each of which is incorporated by reference).Alternatively, or additionally, ADCC activity of the antibody ofinterest may be assessed in vivo, e.g., in an animal model, such as thatdisclosed in Clynes et al, 1998, PNAS USA, vol. 95, page 652, thecontents of which are incorporated by reference in its entirety.

The term “Antibody-dependent cellular phagocytosis” or “ADCP” refers toa process by which antibody-coated cells are internalized, either inwhole or in part, by phagocytic immune cells (e.g., macrophages,neutrophils and dendritic cells) that bind to an immunoglobulin Fcregion.

The term “blood-brain barrier” or “BBB” refers to the physiologicalbarrier between the peripheral circulation and the brain and spinal cordwhich is formed by tight junctions within the brain capillaryendothelial plasma membranes, creating a tight barrier that restrictsthe transport of molecules into the brain, even very small moleculessuch as urea (60 Daltons). The blood-brain barrier within the brain, theblood-spinal cord barrier within the spinal cord, and the blood-retinalbarrier within the retina are contiguous capillary barriers within thecentral nerve system (CNS), and are herein collectively referred to asthe “blood-brain barrier” or “BBB.” The BBB also encompasses theblood-cerebral spinal fluid barrier (choroid plexus) where the barrieris comprised of ependymal cells rather than capillary endothelial cells.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. A “tumor” comprises one or morecancerous cells. Examples of cancer include, but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g., epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer (“NSCLC”),adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, as well as head and neckcancer.

A “comparison window,” as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith (Smith and Waterman, 1981 “Comparison of biosequences”, Adv ApplMath, 2:482-489; Smith and Waterman, 1981, “Overlapping genes andinformation theory”, J Theor Biol, 91:379-380; Smith and Waterman, J MolBiol, “Identification of common molecular subsequences”, 1981,147:195-197; Smith et al., 1981, “”Comparative biosequence metrics”, JMol Evol, 18:38-46), by the homology alignment algorithm of Needleman(Needleman and Wunsch, 1970, “A general method applicable to the searchfor similarities in the amino acid sequence of two proteins” J Mol Biol,48(3):443-453), by the search of similarity method of Pearson (Pearsonand Lipman, 1988, “Improved tools for biological sequence comparison”,Proc Nat Acad Sci USA, 85:2444-2448), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by inspection, and the best alignment(i.e., resulting in the highest percentage of homology over thecomparison window) generated by the various methods is selected.

The term “complement-dependent cytotoxicity (CDC)” refers to a processinitiated by binding of complement factor C1q to the Fc part of most IgGantibody subclasses. Binding of C1q to an antibody is caused by definedprotein-protein interactions at the so called binding site. Such Fc partbinding sites are known in the state of the art (see above). Such Fcpart binding sites are, e.g., characterized by the amino acids L234,L235, D270, N297, E318, K320, K322, P331, and P329 (numbering accordingto EU index of Kabat). Antibodies of subclass IgG1, IgG2, and IgG3usually show complement activation including C1q and C3 binding, whereasIgG4 does not activate the complement system and does not bind C1qand/or C3. C1q is a polypeptide that includes a binding site for the Fcregion of an immunoglobulin. C1q together with two serine proteases, C1rand C1s, forms the complex CI, the first component of the complementdependent cytotoxicity (CDC) pathway.

The term “conditionally active biologic protein” refers to a variant, ormutant, of a wild-type or a parent protein which is more or less activethan the parent or wild-type protein under one or more normalphysiological conditions. This conditionally active protein alsoexhibits activity in selected regions of the body and/or exhibitsincreased or decreased activity under aberrant, or permissive,physiological conditions. Normal physiological conditions are those oftemperature, pH, osmotic pressure, osmolality, oxidation and electrolyteconcentration which would be considered within a normal range at thesite of administration, or at the tissue or organ at the site of action,to a subject. An aberrant condition is that which deviates from thenormally acceptable range for that condition. In one aspect, theconditionally active biologic protein is virtually inactive at wild-typeconditions but is active at other than wild-type conditions at a levelthat is equal or better than at wild-type conditions. For example, inone aspect, an evolved conditionally active biologic protein isvirtually inactive at body temperature, but is active at lowertemperatures. In another aspect, the conditionally active biologicprotein is reversibly or irreversibly inactivated at the wild typeconditions. In a further aspect, the wild-type protein is a therapeuticprotein. In another aspect, the conditionally active biologic protein isused as a drug, or therapeutic agent. In yet another aspect, the proteinis more or less active in highly oxygenated blood, such as, for example,after passage through the lung or in the lower pH environments found inthe kidney.

The term “conditionally active antibody” refers to a variant, or mutant,of a wild-type or parent antibody which is more or less active comparedto the parent or wild-type antibody under one or more normalphysiological conditions. This conditionally active antibody alsoexhibits activity in selected regions of the body and/or exhibitsincreased or decreased activity under aberrant, or permissive,physiological conditions. In one aspect, the conditionally activeantibody is virtually inactive under normal physiological conditions butis active under non-normal physiological conditions at a level that isequal or better than under normal physiological conditions. For example,an evolved conditionally active antibody is virtually inactive at normalbody temperature, but is active at lower body temperatures. In anotheraspect, the conditionally active antibody is reversibly or irreversiblyinactivated under normal physiological conditions. In a further aspect,the wild-type antibody is a therapeutic antibody. In another aspect, theconditionally active antibody is used as a drug, or therapeutic agent.In yet another aspect, the antibody is more or less active in highlyoxygenated blood, for example, after passage through the lung or in thelower pH environments found in the kidney.

“Conservative amino acid substitutions” refer to the interchangeabilityof residues having similar side chains. For example, a group of aminoacids having aliphatic side chains is glycine, alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonessuch as human growth hormones, N-methionyl human growth hormones, andbovine growth hormones; parathyroid hormones; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-a and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-aand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-a, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such asTNF-a or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof. “Digestion” of DNArefers to catalytic cleavage of the DNA with a restriction enzyme thatacts only at certain sequences in the DNA. The various restrictionenzymes used herein are commercially available and their reactionconditions, cofactors and other requirements were used as would be knownto the ordinarily skilled artisan. For analytical purposes, typically 1microgram of plasmid or DNA fragment is used with about 2 units ofenzyme in about 20 microliters of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50micrograms of DNA are digested with 20 to 250 units of enzyme in alarger volume. Appropriate buffers and substrate amounts for particularrestriction enzymes are specified by the manufacturer. Incubation timesof about 1 hour at 37 degrees C. are ordinarily used, but may vary inaccordance with the supplier's instructions. After digestion thereaction is electrophoresed directly on a gel to isolate the desiredfragment.

The term “DNA shuffling” is used herein to indicate recombinationbetween substantially homologous but non-identical sequences, in someembodiments DNA shuffling may involve crossover via non-homologousrecombination, such as via cer/lox and/or flp/frt systems and the like.DNA shuffling can be random or non-random.

The term “drug” or “drug molecule” refers to a therapeutic agentincluding a substance having a beneficial effect on a human or animalbody when it is administered to the human or animal body. Preferably,the therapeutic agent includes a substance that can treat, cure orrelieve one or more symptoms, illnesses, or abnormal conditions in ahuman or animal body or enhance the wellness of a human or animal body.

An “effective amount” is an amount of a conditionally active biologicprotein or fragment which is effective to treat or prevent a conditionin a living organism to whom it is administered over some period oftime, e.g., provides a therapeutic effect during a desired dosinginterval.

As used herein, the term “electrolyte” is used to define a mineral inthe blood or other body fluids that carries a charge. For example, inone aspect, the normal physiological condition and aberrant conditioncan be conditions of “electrolyte concentration”. In one aspect, theelectrolyte concentration to be tested is selected from one or more ofionized calcium, sodium, potassium, magnesium, chloride, bicarbonate,and phosphate concentration. For example, in one aspect, normal range ofserum calcium is 8.5 to 10.2 mg/dL. In this aspect, aberrant serumcalcium concentration may be selected from either above or below thenormal range, m another example, in one aspect, normal range of serumchloride is 96-106 milliequivalents per liter (mEq/L). In this aspect,aberrant serum chloride concentration may be selected from either aboveor below the normal range, in another example, in one aspect, a normalrange of serum magnesium is from 1.7-2.2 mg/dL. In this aspect, anaberrant serum magnesium concentration may be selected from either aboveor below the normal range, in another example, in one aspect, a normalrange of serum phosphorus is from 2.4 to 4.1 mg/dL. In this aspect,aberrant serum phosphorus concentration may be selected from eitherabove or below the normal range. In another example, in one aspect, anormal range of serum, or blood, sodium is from 135 to 145 mEq/L. Inthis aspect, aberrant serum, or blood, sodium concentration may beselected from either above or below the normal range. In anotherexample, in one aspect, a normal range of serum, or blood, potassium isfrom 3.7 to 5.2 mEq/L. In this aspect, aberrant serum, or blood,potassium concentration maybe selected from either above or below thenormal range. In a further aspect, a normal range of serum bicarbonateis from 20 to 29 mEq/L. In this aspect, aberrant serum, or blood,bicarbonate concentration may be selected from either above or below thenormal range. In a different aspect, bicarbonate levels can be used toindicate normal levels of acidity (pH), in the blood. The term“electrolyte concentration” may also be used to define the condition ofa particular electrolyte in a tissue or body fluid other than blood orplasma. In this case, the normal physiological condition is consideredto be the clinically normal range for that tissue or fluid. In thisaspect, aberrant tissue or fluid electrolyte concentration may beselected from either above or below the normal range.

As used in this disclosure, the term “epitope” refers to an antigenicdeterminant on an antigen, such as an enzyme polypeptide, to which theparatope of an antibody, such as an enzyme-specific antibody, binds.Antigenic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three-dimensional structural characteristics, as wellas specific charge characteristics. As used herein “epitope” refers tothat portion of an antigen or other macromolecule capable of forming abinding interaction that interacts with the variable region binding bodyof an antibody. Typically, such binding interaction is manifested as anintermolecular contact with one or more amino acid residues of a CDR.

As used herein, an “enzyme” is a protein with specific catalyticproperties. Factors such as, for example, substrate concentration, pH,temperature and presence or absence of inhibitors can affect the rate ofcatalysis. Typically, for a wild type enzyme, Q10 (the temperaturecoefficient) describes the increase in reaction rate with a 10 degree C.rise in temperature. For wild type enzymes, the Q10=2 to 3; in otherwords, the rate of reaction doubles or triples with every 10 degreeincrease in temperature. At high temperatures, proteins denature. At pHvalues slightly different from an enzymes optimum value, small changesoccur in the charges of the enzyme and perhaps the substrate molecule.The change in ionization can affect the binding of the substratemolecule. At extreme pH levels, the enzyme will produce denaturation,where the active site is distorted, and the substrate molecule will nolonger fit.

As used herein, the term “evolution”, or “evolving”, refers to using oneor more methods of mutagenesis to generate a novel polynucleotideencoding a novel polypeptide, which novel polypeptide is itself animproved biological molecule &/or contributes to the generation ofanother improved biological molecule. In a particular non-limitingaspect, the present disclosure relates to evolution of conditionallyactive biologic proteins from a parent wild type protein. In one aspect,for example, evolution relates to a method of performing bothnon-stochastic polynucleotide chimerization and non-stochasticsite-directed point mutagenesis disclosed in U.S. patent applicationpublication 2009/0130718, which is incorporated herein by reference.More particularly, the present disclosure provides methods for evolutionof conditionally active biologic enzymes which exhibit reduced activityat normal physiological conditions compared to a wild-type enzyme parentmolecule, but enhanced activity under one or more aberrant conditionscompared to the wild-type enzyme.

The terms “fragment”, “derivative” and “analog” when referring to areference polypeptide comprise a polypeptide which retains at least onebiological function or activity that is at least essentially same asthat of the reference polypeptide. Furthermore, the terms “fragment”,“derivative” or “analog” are exemplified by a “pro-form” molecule, suchas a low activity proprotein that can be modified by cleavage to producea mature enzyme with significantly higher activity.

The term “full length antibody” refers to an antibody which comprises anantigen-binding variable region (VH or VL) as well as a light chainconstant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3.The constant domains may be native sequence constant domains (e.g. humannative sequence constant domains) or amino acid sequence variantsthereof.

Depending on the amino acid sequence of the constant domain of theirheavy chains, full length antibodies can be assigned to different“classes”. There are five major classes of full length antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

A method is provided herein for producing from a template polypeptide aset of progeny polypeptides in which a “full range of single amino acidsubstitutions” is represented at each amino acid position. As usedherein, “full range of single amino acid substitutions” is in referenceto the 20 naturally encoded polypeptide-forming alpha-amino acids, asdescribed herein.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(nitrons) between individual coding segments (exons).

“Genetic instability”, as used herein, refers to the natural tendency ofhighly repetitive sequences to be lost through a process of reductiveevents generally involving sequence simplification through the loss ofrepeated sequences. Deletions tend to involve the loss of one copy of arepeat and everything between the repeats.

The term “growth factor” refers to proteins that promote growth, andinclude, for example, hepatic growth factors; fibroblast growth factors;vascular endothelial growth factors; nerve growth factors such as NGF-β;platelet-derived growth factors; transforming growth factors (TGFs) suchas TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin(EPO); osteoinductive factors; interferons such as interferon-α, -β, and-γ, and colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF). As used herein, the term growth factor includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native-sequence growth factor, includingsynthetically produced small-molecule entities and pharmaceuticallyacceptable derivatives and salts thereof.

The term “heterologous” means that one single-stranded nucleic acidsequence is unable to hybridize to another single-stranded nucleic acidsequence or its complement. Thus areas of heterology means that areas ofpolynucleotides or polynucleotides have areas or regions within theirsequence which are unable to hybridize to another nucleic acid orpolynucleotide. Such regions or areas are for example areas ofmutations.

The term “hormone” refers to polypeptide hormones, which are generallysecreted by glandular organs with ducts. Included among the hormonesare, for example, growth hormones such as human growth hormones,N-methionyl human growth hormones, and bovine growth hormones;parathyroid hormones; thyroxine; insulin; proinsulin; relaxin;estradiol; hormone-replacement therapy; androgens such as calusterone,dromostanolone propionate, epitiostanol, mepitiostane, or testolactone;prorelaxin; glycoprotein hormones such as follicle stimulating hormones(FSH), thyroid stimulating hormones (TSH), and luteinizing hormones(LH); prolactin, placental lactogen, mouse gonadotropin-associatedpeptide, gonadotropin-releasing hormones; inhibin; activin;mullerian-inhibiting substance; and thrombopoietm. As used herein, theterm hormone includes proteins from natural sources or from recombinantcell culture and biologically active equivalents of the native-sequencehormone, including synthetically produced small-molecule entities andpharmaceutically acceptable derivatives and salts thereof.

The term “identical” or “identity” means that two nucleic acid sequenceshave the same sequence or a complementary sequence. Thus, “areas ofidentity” means that regions or areas of a polynucleotide or the overallpolynucleotide are identical or complementary to areas of anotherpolynucleotide.

The term “immune system checkpoint,” or “immune checkpoint”, refers toone or more inhibitory pathways in the immune system that contribute tothe maintenance of self-tolerance or modulation of the duration andamplitude of physiological immune responses to minimize collateraltissue damages. The immune checkpoint functions as a safeguard forpreventing the immune system from attacking host molecules or cells(self-tolerance). When the immune checkpoints are inhibited, the immunesystem, especially the T-cells, becomes super activated, which may leadto a loss of self-tolerance. The loss of self-tolerance may result inhost molecules, cells, and/or tissues being attacked by the immunesystem thereby causing collateral tissue damage, in addition toattacking foreign molecules or cells. When these immune checkpoints arenot inhibited, the immune system can achieve a balance betweenself-tolerance and attacking foreign molecules and cells in the body.

It has been found that tumor tissue and possibly certain pathogens havethe ability to cope with the immune checkpoints to reduce theeffectiveness of host immune response, resulting in tumor growth and/orchronic infection (see, e.g., Pardoll, Nature Reviews Cancer, vol. 12,pages 252-264, 2012; Nirschl & Drake, Clin Cancer Res, vol. 19, pages4917-4924, 2013). However, a super-activated immune system initiated byinhibition of the immune checkpoints is much more sensitive and thus candetect and attack tumors. Thus, for cancer therapy, immune checkpointinhibition is a desirable goal in order to allow the immune system toparticipate in the fight against tumors. The problem that must beaddressed is how to super-activate the immune system to fight tumors,while minimizing the potential for collateral damage to other parts ofthe body.

The term “immune checkpoint inhibitor” as used herein refers tomolecules that totally or partially reduce, inhibit, interfere with ormodulate one or more immune checkpoint proteins. Immune checkpointproteins regulate the immune system, especially T-cells, activation orfunction. Numerous immune checkpoint proteins are known, such ascytotoxic T-lymphocyte antigen 4 (CTLA4) and its ligands CD 80 and CD86,and programmed cell death 1 protein (PD1) and its ligands PDL1 and PDL2(Pardoll, Nature Reviews Cancer, vol. 12, pages 252-264, 2012). Theseproteins are responsible for interactions that inhibit T-cell responses.Immune checkpoint proteins regulate and maintain self-tolerance, as wellas the duration and amplitude of physiological immune responses. Immunecheckpoint inhibitors may include antibodies or may be derived fromantibodies. For example, antibodies that bind to CTLA4, PD-1, or PD-L1function as immune checkpoint inhibitors.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide or enzymepresent in a living animal is not isolated, but the same polynucleotideor enzyme, separated from some or all of the coexisting materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or enzymes could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

The term “isolated nucleic acid” is used to define a nucleic acid, e.g.,a DNA or RNA molecule, that is not immediately contiguous with the 5′and 3′ flanking sequences with which it normally is immediatelycontiguous when present in the naturally occurring genome of theorganism from which it is derived. The term thus describes, for example,a nucleic acid that is incorporated into a vector, such as a plasmid orviral vector; a nucleic acid that is incorporated into the genome of aheterologous cell (or the genome of a homologous cell, but at a sitedifferent from that at which it naturally occurs); and a nucleic acidthat exists as a separate molecule, e.g., a DNA fragment produced by PCRamplification or restriction enzyme digestion, or an RNA moleculeproduced by in vitro transcription. The term also describes arecombinant nucleic acid that forms part of a hybrid gene encodingadditional polypeptide sequences that can be used, for example, in theproduction of a fusion protein.

The term “joint damage” is used in the broadest sense and refers to anydamage or partial or complete destruction to any part of one or morejoints, including the connective tissue and cartilage, where damageincludes structural and/or functional damage of any cause, and may ormay not cause joint pain/arthalgia. It includes, without limitation,joint damage associated with or resulting from inflammatory jointdisease as well as non-inflammatory joint disease. This damage may becaused by any condition, such as an autoimmune disease such as lupus(e.g., systemic lupus erythematosus), arthritis (e.g., acute and chronicarthritis, rheumatoid arthritis (RA) including juvenile-onset rheumatoidarthritis, juvenile idiopathic arthritis (JIA), or juvenile RA (JRA)).Other conditions and diseases include rheumatoid synovitis, gout orgouty arthritis, acute immunological arthritis, chronic inflammatoryarthritis, degenerative arthritis, type II collagen-induced arthritis,infectious arthritis, septic arthritis, Lyme arthritis, proliferativearthritis, psoriatic arthritis, Still's disease, vertebral arthritis,osteoarthritis, arthritis chronica progrediente, arthritis deformans,polyarthritis chronica primaria, reactive arthritis, menopausalarthritis, estrogen-depletion arthritis, and ankylosingspondylitis/rheumatoid spondylitis), rheumatic autoimmune disease otherthan RA, significant systemic involvement secondary to RA (including butnot limited to vasculitis, pulmonary fibrosis or Felty's syndrome),Sjogren's syndrome, particular secondary such syndrome. Furtherconditions include secondary limited cutaneous vasculitis with RA,seronegative spondyloarthropathy, Lyme disease, inflammatory boweldisease, scleroderma, inflammatory myopathy, mixed connective tissuedisease, any overlap syndrome, bursitis, tendonitis, osteomyelitis,infectious diseases, including influenza, measles (rubeola), rheumaticfever, Epstein-Barr viral syndrome, hepatitis, mumps, rebella (Germanmeasles), and varicella (chickenpox), Chondromalacia patellae,collagenous colitis, autoimmune disorders associated with collagendisease, joint inflammation, unusual exertion or overuse such as sprainsor strains, injury including fracture, gout, especially found in the bigtoe, as well as caused by neurological disorders, hemophilic disorders(for example, hemophilic arthropathy), muscular disorders, progressivedisorders, bone disorders, cartilage disorders, and vascular disorders.For purposes herein, joints are points of contact between elements of askeleton (of a vertebrate such as an animal) with the parts thatsurround and support it include, but are not limited to, hips, jointsbetween the vertebrae of the spine, joints between the spine and pelvis(sacroiliac joints), joints where the tendons and ligaments attach tobones, joints between the ribs and spine, shoulders, knees, feet,elbows, hands, fingers, ankles and toes, but especially joints in thehands and feet.

As used herein “ligand” refers to a molecule, such as a random peptideor variable segment sequence, that is recognized by a particularreceptor. As one of skill in the art will recognize, a molecule (ormacromolecular complex) can be both a receptor and a ligand. In general,the binding partner having a smaller molecular weight is referred to asthe ligand and the binding partner having a greater molecular weight isreferred to as a receptor.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Sambrook et al., (1982).Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory,Cold Spring Harbor, N.Y., p. 146; Sambrook et al., Molecular Cloning: alaboratory manual, 2^(nd) Ed., Cold Spring Harbor Laboratory Press,1989). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 micrograms of approximately equimolar amounts of the DNAfragments to be ligated.

As used herein, “linker” or “spacer” refers to a molecule or group ofmolecules that connects two molecules, such as a DNA binding protein anda random peptide, and serves to place the two molecules in a preferredconfiguration, e.g., so that the random peptide can bind to a receptorwith minimal steric hindrance from the DNA binding protein.

The term “mammalian cell surface display” refers to a technique wherebya protein or antibody, or a portion of an antibody, is expressed anddisplayed on a mammalian host cell surface for screening purposes; forexample, by screening for specific antigen binding by a combination ofmagnetic beads and fluorescence-activated cell sorting. In one aspect,mammalian expression vectors are used for simultaneous expression ofimmunoglobulins as both a secreted and cell surface bound form as inDuBridge et al., US 2009/0136950, which is incorporated herein byreference for the disclosure of this aspect. In another aspect, thetechniques are employed for a viral vector encoding for a library ofantibodies or antibody fragments are displayed on the cell membraneswhen expressed in a cell as in Gao et al., US 2007/0111260, incorporatedherein by reference for the disclosure of this aspect.

Whole IgG surface display on mammalian cells is known. For example,Akamatsuu et al. developed a mammalian cell surface display vector,suitable for directly isolating IgG molecules based on theirantigen-binding affinity and biological activity. Using an Epstein-Barrvirus-derived episomal vector, antibody libraries were displayed aswhole IgG molecules on the cell surface and screened for specificantigen binding by a combination of magnetic beads andfluorescence-activated cell sorting. Plasmids encoding antibodies withdesired binding characteristics were recovered from sorted cells andconverted to the form for production of soluble IgG. See Akamatsuu etal. J. Immunol. Methods, vol. 327, pages 40-52, 2007, incorporatedherein by reference. Ho et al. used human embryonic kidney 293T cellsthat are widely used for transient protein expression for cell surfacedisplay of single-chain Fv antibodies for affinity maturation. Cellsexpressing a rare mutant antibody with higher affinity were enriched240-fold by a single-pass cell sorting from a large excess of cellsexpressing WT antibody with a slightly lower affinity. Furthermore, ahighly enriched mutant was obtained with increased binding affinity forCD22 after a single selection of a combinatory library randomizing anintrinsic antibody hotspot. See Ho et al., “Isolation of anti-CD22 Fvwith high affinity by Fv display on human cells,” Proc Natl Acad SciUSA, vol. 103, pages 9637-9642, 2006, incorporated herein by reference.

B cells specific for an antigen can also be used. Such B cells weredirectly isolated from peripheral blood mononuclear cells (PBMC) ofhuman donors. Recombinant, antigen-specific single-chain Fv (scFv)libraries are generated from this pool of B cells and screened bymammalian cell surface display by using a Sindbis virus expressionsystem. This method allows isolating antigen-specific antibodies by asingle round of FACS. The variable regions (VRs) of the heavy chains(HCs) and light chains (LCs) were isolated from positive clones andrecombinant fully human antibodies produced as whole IgG or Fabfragments. In this manner, several hypermutated high-affinity antibodiesbinding the Qβ virus like particle (VLP), a model viral antigen, as wellas antibodies specific for nicotine were isolated. All antibodies showedhigh expression levels in cell culture. The human nicotine-specific mAbswere validated preclinically in a mouse model. See Beerli et al.,“Isolation of human monoclonal antibodies by mammalian cell display,”Proc Natl Acad Sci USA, vol. 105, pages 14336-14341, 2008, incorporatedherein by reference.

Yeast cell surface display may also be used in the present invention,for example, see Kondo and Ueda, “Yeast cell-surfacedisplay-applications of molecular display,” Appl. Microbiol.Biotechnol., vol. 64, pages 28-40, 2004, which describes for example, acell-surface engineering system using the yeast Saccharomycescerevisiae. Several representative display systems for the expression inyeast S. cerevisiae are described in Lee et al, “Microbial cell-surfacedisplay,” TRENDS in Bitechnol., vol. 21, pages 45-52, 2003. Also Boderand Wittrup, “Yeast surface display for screening combinatorialpolypeptide libraries,” Nature Biotechnol., vol. 15, pages 553, 1997.

As used herein “microenvironment” means any portion or region of atissue or body that has constant or temporal, physical or chemicaldifferences from other regions of the tissue or regions of the body.

As used herein, a “molecular property to be evolved” includes referenceto molecules comprised of a polynucleotide sequence, molecules comprisedof a polypeptide sequence, and molecules comprised in part of apolynucleotide sequence and in part of a polypeptide sequence.Particularly relevant—but by no means limiting—examples of molecularproperties to be evolved include protein activities at specifiedconditions, such as related to temperature; salinity; osmotic pressure;pH; oxidation, and concentration of glycerol, DMSO, detergent, &/or anyother molecular species with which contact is made in a reactionenvironment. Additional particularly relevant—but by no meanslimiting—examples of molecular properties to be evolved includestabilities—e.g. the amount of a residual molecular property that ispresent after a specified exposure time to a specified environment, suchas may be encountered during storage.

The term “multispecific antibody” as used herein is an antibody havingbinding specificities for at least two different epitopes. Exemplarymultispecific antibodies may bind both a BBB-R and a brain antigen.Multispecific antibodies can be prepared as full-length antibodies orantibody fragments (e.g. F(ab′)2 bispecific antibodies). Engineeredantibodies binding two, three or more (e.g. four) antigens arecontemplated (see, e.g., US 2002/0004587 A1). One or more wild-typeantibody(s) may be engineered to be multispecific, or two antibodies maybe engineered to comprise a multispecific antibody. Multispecificantibodies can be multifunctional.

The term “mutations” means changes in the sequence of a wild-typenucleic acid sequence or changes in the sequence of a peptide. Suchmutations may be point mutations such as transitions or transversions.The mutations may be deletions, insertions or duplications.

As used herein, the degenerate “N,N,G/T” nucleotide sequence represents32 possible triplets, where “N” can be A, C, G or T.

The term “naturally-occurring” as used herein as applied to the objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring. Generally, the term naturally occurring refers toan object as present in a non-pathological (un-diseased) individual,such as would be typical for the species.

As used herein, “normal physiological conditions”, or “wild typeoperating conditions”, are those conditions of temperature, pH, osmoticpressure, osmolality, oxidation and electrolyte concentration whichwould be considered within a normal range at the site of administration,or the site of action, in a subject.

As used herein, a “nucleic acid molecule” is comprised of at least onebase or one base pair, depending on whether it is single-stranded ordouble-stranded, respectively. Furthermore, a nucleic acid molecule maybelong exclusively or chimerically to any group of nucleotide-containingmolecules, as exemplified by, but not limited to, the following groupsof nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomicnucleic acids, naturally occurring and not naturally occurring nucleicacids, and synthetic nucleic acids. This includes, by way ofnon-limiting example, nucleic acids associated with any organelle, suchas the mitochondria, ribosomal RNA, and nucleic acid molecules comprisedchimerically of one or more components that are not naturally occurringalong with naturally occurring components.

Additionally, a “nucleic acid molecule” may contain in part one or morenon-nucleotide-based components as exemplified by, but not limited to,amino acids and sugars. Thus, by way of example, but not limitation, aribozyme that is in part nucleotide-based and in part protein-based isconsidered a “nucleic acid molecule”.

In addition, by way of example, but not limitation, a nucleic acidmolecule that is labeled with a detectable moiety, such as a radioactiveor alternatively a nonradioactive label, is likewise considered a“nucleic acid molecule”.

he terms “nucleic acid sequence coding for” or a “DNA coding sequence ofor a “nucleotide sequence encoding” a particular enzyme—as well as othersynonymous terms—refer to a DNA sequence which is transcribed andtranslated into an enzyme when placed under the control of appropriateregulatory sequences. A “promotor sequence” is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3′ direction) coding sequence. The promoter is part ofthe DNA sequence. This sequence region has a start codon at its 3′terminus. The promoter sequence does include the minimum number of baseswhere elements necessary to initiate transcription at levels detectableabove background. However, after the RNA polymerase binds the sequenceand transcription is initiated at the start codon (3′ terminus with apromoter), transcription proceeds downstream in the 3′ direction. Withinthe promoter sequence will be found a transcription initiation site(conveniently defined by mapping with nuclease S1) as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The terms “nucleic acid encoding an enzyme (protein)” or “DNA encodingan enzyme (protein)” or “polynucleotide encoding an enzyme (protein)”and other synonymous terms encompasses a polynucleotide which includesonly coding sequence for the enzyme as well as a polynucleotide whichincludes additional coding and/or non-coding sequence.

In one preferred embodiment, a “specific nucleic acid molecule species”is defined by its chemical structure, as exemplified by, but not limitedto, its primary sequence. In another preferred embodiment, a specific“nucleic acid molecule species” is defined by a function of the nucleicacid species or by a function of a product derived from the nucleic acidspecies. Thus, by way of non-limiting example, a “specific nucleic acidmolecule species” may be defined by one or more activities or propertiesattributable to it, including activities or properties attributable toits expressed product.

The instant definition of “assembling a working nucleic acid sample intoa nucleic acid library” includes the process of incorporating a nucleicacid sample into a vector-based collection, such as by ligation into avector and transformation of a host. A description of relevant vectors,hosts, and other reagents as well as specific non-limiting examplesthereof are provided hereinafter. The instant definition of “assemblinga working nucleic acid sample into a nucleic acid library” also includesthe process of incorporating a nucleic acid sample into anon-vector-based collection, such as by ligation to adaptors. Preferablythe adaptors can anneal to PCR primers to facilitate amplification byPCR.

Accordingly, in a non-limiting embodiment, a “nucleic acid library” iscomprised of a vector-based collection of one or more nucleic acidmolecules. In another preferred embodiment a “nucleic acid library” iscomprised of a non-vector-based collection of nucleic acid molecules. Inyet another preferred embodiment a “nucleic acid library” is comprisedof a combined collection of nucleic acid molecules that is in partvector-based and in part non-vector-based. Preferably, the collection ofmolecules comprising a library is searchable and separable according toindividual nucleic acid molecule species.

The present disclosure provides a “nucleic acid construct” oralternatively a “nucleotide construct” or alternatively a “DNAconstruct”. The term “construct” is used herein to describe a molecule,such as a polynucleotide (e.g., an enzyme polynucleotide) which mayoptionally be chemically bonded to one or more additional molecularmoieties, such as a vector, or parts of a vector. In a specific—but byno means limiting—aspect, a nucleotide construct is exemplified by DNAexpression constructs suitable for the transformation of a host cell.

An “oligonucleotide” (or synonymously an “oligo”) refers to either asingle stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands which may be chemically synthesized. Suchsynthetic oligonucleotides may or may not have a 5′ phosphate. Thosethat do not will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated. To achieve polymerase-based amplification (such aswith PCR), a “32-fold degenerate oligonucleotide that is comprised of,in series, at least a first homologous sequence, a degenerate N,N,G/Tsequence, and a second homologous sequence” is mentioned. As used inthis context, “homologous” is in reference to homology between the oligoand the parental polynucleotide that is subjected to thepolymerase-based amplification.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame.

A coding sequence is “operably linked to” another coding sequence whenRNA polymerase will transcribe the two coding sequences into a singlemRNA, which is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein the term “parental polynucleotide set” is a set comprisedof one or more distinct polynucleotide species. Usually this term isused in reference to a progeny polynucleotide set which is preferablyobtained by mutagenization of the parental set, in which case the terms“parental”, “starting” and “template” are used interchangeably.

The term “patient”, “individual” or “subject”, refers to an animal, forexample a mammal, such as a human, who is the object of treatment. Thesubject, or patient, may be either male or female. Mammals include, butare not limited to, domesticated animals (e.g., cows, sheep, cats, dogs,and horses), primates (e.g., humans and non-human primates such asmonkeys), rabbits, and rodents (e.g., mice and rats). In certainembodiments, the individual or subject is a human.

As used herein the term “physiological conditions” refers totemperature, pH, osmotic pressure, ionic strength, viscosity, and likebiochemical parameters which are compatible with a viable organism,and/or which typically exist intracellularly in a viable cultured yeastcell or mammalian cell. For example, the intracellular conditions in ayeast cell grown under typical laboratory culture conditions arephysiological conditions. Suitable in vitro reaction conditions for invitro transcription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions comprise 50-200 mM NaCl orKCl, pH 6.5-8.5, 20-45 degrees C. and 0.001-10 mM divalent cation (e.g.,Mg^(++″), Ca⁺⁺); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mMdivalent cation, and often include 0.01-1.0 percent nonspecific protein(e.g., BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) canoften be present, usually at about 0.001 to 2%, typically 0.05-0.2%(v/v). Particular aqueous conditions may be selected by the practitioneraccording to conventional methods. For general guidance, the followingbuffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mMTris HCl, pH 5-8, with optional addition of divalent cation(s) and/ormetal chelators and/or non-ionic detergents and/or membrane fractionsand/or anti-foam agents and/or scintillants. Normal physiologicalconditions refer to conditions of temperature, pH, osmotic pressure,osmolality, oxidation and electrolyte concentration in vivo in a patientor subject at the site of administration, or the site of action, whichwould be considered within the normal range in a patient.

Standard convention (5′ to 3′) is used herein to describe the sequenceof double stranded polynucleotides.

The term “polyepitopic specificity” refers to the ability of amultispecific or multifunctional antibody to specifically bind to two ormore different epitopes on the same target or on different targets.

The term “epitope” refers to a specific amino acid sequence, modifiedamino acid sequence, or protein secondary or tertiary structure which isrecognized by an antibody.

The term “population” as used herein means a collection of componentssuch as polynucleotides, portions or polynucleotides or proteins. A“mixed population” means a collection of components which belong to thesame family of nucleic acids or proteins (i.e., are related) but whichdiffer in their sequence (i.e., are not identical) and hence in theirbiological activity.

A molecule having a “pro-form” refers to a molecule that undergoes anycombination of one or more covalent and noncovalent chemicalmodifications (e.g. glycosylation, proteolytic cleavage, dimerization oroligomerization, temperature-induced or pH-induced conformationalchange, association with a co-factor, etc.) en route to attain a moremature molecular form having a property difference (e.g. an increase inactivity) in comparison with the reference pro-form molecule. When twoor more chemical modifications (e.g. two proteolytic cleavages, or aproteolytic cleavage and a deglycosylation) can be distinguished enroute to the production of a mature molecule, the reference precursormolecule may be termed a “pre-pro-form” molecule.

As used herein, the term “pseudorandom” refers to a set of sequencesthat have limited variability, such that, for example, the degree ofresidue variability at another position, but any pseudorandom positionis allowed some degree of residue variation, however circumscribed.

“Quasi-repeated units”, as used herein, refers to the repeats to bere-assorted and are by definition not identical. Indeed the method isproposed not only for practically identical encoding units produced bymutagenesis of the identical starting sequence, but also thereassortment of similar or related sequences which may divergesignificantly in some regions. Nevertheless, if the sequences containsufficient homologies to be reasserted by this approach, they can bereferred to as “quasi-repeated” units.

As used herein, “receptor” refers to a molecule that has an affinity fora given ligand. Receptors can be naturally occurring or syntheticmolecules. Receptors can be employed in an unaltered state or asaggregates with other species. Receptors can be attached, covalently ornon-covalently, to a binding member, either directly or via a specificbinding substance. Examples of receptors include, but are not limitedto, antibodies, including monoclonal antibodies and antisera reactivewith specific antigenic determinants (such as on viruses, cells, orother materials), cell membrane receptors, complex carbohydrates andglycoproteins, enzymes, and hormone receptors.

The term “recombinant antibody”, as used herein, refers to an antibody(e.g. a chimeric, humanized, or human antibody or antigen-bindingfragment thereof) that is expressed by a recombinant host cellcomprising nucleic acid encoding the antibody. Examples of “host cells”for producing recombinant antibodies include: (1) mammalian cells, forexample, Chinese Hamster Ovary (CHO), COS, myeloma cells (including Y0and NS0 cells), baby hamster kidney (BHK), Hela and Vero cells; (2)insect cells, for example, sf9, sf21 and Tn5; (3) plant cells, forexample plants belonging to the genus Nicotiana (e.g. Nicotianatabacum); (4) yeast cells, for example, those belonging to the genusSaccharomyces (e.g. Saccharomyces cerevisiae) or the genus Aspergillus(e.g. Aspergillus niger); (5) bacterial cells, for example Escherichia.coli cells or Bacillus subtilis cells, etc.

“Recombinant” enzymes refer to enzymes produced by recombinant DNAtechniques, i.e., produced from cells transformed by an exogenous DNAconstruct encoding the desired enzyme. “Synthetic” enzymes are thoseprepared by chemical synthesis.

“Reductive reassortment”, as used herein, refers to the increase inmolecular diversity that is accrued through deletion (and/or insertion)events that are mediated by repeated sequences.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence,” “comparisonwindow,” “sequence identity,” “percentage of sequence identity,” and“substantial identity.”

A “reference sequence” is a defined sequence used as a basis for asequence comparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence given in a sequence listing, or may comprise a complete cDNA orgene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the two polynucleotidesand (2) may further comprise a sequence that is divergent between thetwo polynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity.

“Repetitive Index (RI)”, as used herein, is the average number of copiesof the quasi-repeated units contained in the cloning vector.

The term “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. This “substantial identity”, as used herein,denotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence having at least 80 percent sequenceidentity, preferably at least 85 percent identity, often 90 to 95percent sequence identity, and most commonly at least 99 percentsequence identity as compared to a reference sequence of a comparisonwindow of at least 25-50 nucleotides, wherein the percentage of sequenceidentity is calculated by comparing the reference sequence to thepolynucleotide sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the window ofcomparison.

As known in the art “similarity” between two enzymes is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one enzyme to the sequence of a second enzyme. Similaritymay be determined by procedures which are well-known in the art, forexample, a BLAST program (Basic Local Alignment Search Tool at theNational Center for Biological Information).

As used herein, the term “single-chain antibody” refers to a polypeptidecomprising a VH domain and a VL domain in polypeptide linkage, generallyliked via a spacer peptide, and which may comprise additional amino acidsequences at the amino- and/or carboxy-termini. For example, asingle-chain antibody may comprise a tether segment for linking to theencoding polynucleotide. As an example a scFv is a single-chainantibody. Single-chain antibodies are generally proteins consisting ofone or more polypeptide segments of at least 10 contiguous aminosubstantially encoded by genes of the immunoglobulin superfamily (e.g,see The Immunoglobulin Gene Superfamily, A. F. Williams and A. N.Barclay, in Immunoglobulin Genes, T. Honjo, F. W. Alt, and T. H.Rabbits, eds., (1989) Academic press: San Diego, Calif., pp. 361-368,which is incorporated herein by reference), most frequently encoded by arodent, non-human primate, avian, porcine bovine, ovine, goat, or humanheavy chain or light chain gene sequence. A functional single-chainantibody generally contains a sufficient portion of an immunoglobulinsuperfamily gene product so as to retain the property of binding to aspecific target molecule, typically a receptor or antigen (epitope).

The members of a pair of molecules (e.g., an antibody-antigen pair or anucleic acid pair) are said to “specifically bind” to each other if theybind to each other with greater affinity than to other, non-specificmolecules. For example, an antibody raised against an antigen to whichit binds more efficiently than to a non-specific protein can bedescribed as specifically binding to the antigen. (Similarly, a nucleicacid probe can be described as specifically binding to a nucleic acidtarget if it forms a specific duplex with the target by base pairinginteractions (see above).)

“Specific hybridization” is defined herein as the formation of hybridsbetween a first polynucleotide and a second polynucleotide (e.g., apolynucleotide having a distinct but substantially identical sequence tothe first polynucleotide), wherein substantially unrelatedpolynucleotide sequences do not form hybrids in the mixture.

The term “treating” includes: (1) preventing or delaying the appearanceof clinical symptoms of the state, disorder or condition developing inan animal that may be afflicted with or predisposed to the state,disorder or condition but does not yet experience or display clinical orsubclinical symptoms of the state, disorder or condition; (2) inhibitingthe state, disorder or condition (i.e., arresting, reducing or delayingthe development of the disease, or a relapse thereof in case ofmaintenance treatment, of at least one clinical or subclinical symptomthereof); and/or (3) relieving the condition (i.e., causing regressionof the state, disorder or condition or at least one of its clinical orsubclinical symptoms). The benefit to a patient to be treated is eitherstatistically significant or at least perceptible to the patient or tothe physician.

The term “variant” refers to polynucleotides or polypeptides of thedisclosure modified at one or more base pairs, codons, introns, exons,or amino acid residues (respectively) of a wild-type protein parentmolecule. Variants can be produced by any number of means includingmethods such as, for example, error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, saturation mutagenesis and any combinationthereof. Techniques for producing variant proteins having reducedactivity compared to the wild-type protein at a normal physiologicalcondition of e.g., one or more conditions of temperature, pH, osmoticpressure, osmolality, oxidation and electrolyte concentration; andenhanced activity at an aberrant condition, are disclosed herein.Variants may additionally be selected for the properties of enhancedchemical resistance, and proteolytic resistance, compared to thewild-type protein.

As used herein, the term “wild-type” means that the polynucleotide doesnot comprise any mutations, and includes a template protein used as aparent molecule for evolution or other engineering. The “wild-typeprotein” preferably has some desired properties, such as higher bindingaffinity, or enzymatic activity, which may be obtained by screening of alibrary of proteins for a desired properties, including better stabilityin different temperature or pH environments, or improved selectivityand/or solubility. A “wild type protein”, “wild-type protein”,“wild-type biologic protein”, or “wild type biologic protein”, refers toa protein which can be isolated from nature that will be active at alevel of activity found in nature and will comprise the amino acidsequence found in nature. The terms “parent molecule”, “target protein”and “template” can also refer to the wild-type protein.

DETAILED DESCRIPTION

The present disclosure is directed to methods of engineering or evolvingproteins to generate new molecules that are reversibly or irreversiblyinactivated at the wild type condition, but active at non-normalconditions at the same or equivalent level as the activity at thewild-type condition. These new proteins are referred to as conditionallyactive proteins herein. Methods of producing these proteins have beendescribed in US 2012/0164127, which is incorporated herein by referencein its entirety. Conditionally active proteins are particularly valuablefor development of novel therapeutics that are active for short orlimited periods of time within the host. This is particularly valuablewhere extended operation of the protein at the given dose would beharmful to the host, but where limited activity is required to performthe desired therapy. Examples of beneficial applications include topicalor systemic treatments at high dose, as well as localized treatments inhigh concentration. Inactivation under the physiological condition canbe determined by a combination of the dosing and the rate ofinactivation of the protein. This condition based inactivation isespecially important for enzyme therapeutics where catalytic activitycause substantial negative effects in a relatively short period of time.

The present disclosure is also directed to methods of engineering orevolving proteins to generate new molecules that are different from wildtype molecules in that they are reversibly or irreversibly activated orinactivated over time, or activated or inactivated only when they are incertain microenvironments in the body, including in specific organs inthe body. In some embodiments, the conditionally active proteins areantibodies against a suitable antigen.

Target Wild-Type Proteins

Any therapeutic protein can serve as a target protein, or wild-typeprotein, for production of a conditionally active biologic protein. Inone aspect, the target protein is a wild-type enzyme. Currently usedtherapeutic enzymes include urokinase and streptokinase, used in thetreatment of blood clots; and hyaluronidase, used as an adjuvant toimprove the absorption and dispersion of other drugs, in one aspect, thewild-type protein selected for generation of a conditionally activebiologic protein can be a currently used therapeutic enzyme, in order toavoid or minimize deleterious side effects associated with the wild-typeprotein or enzyme. Alternatively, an enzyme not in current usage as atherapeutic can be selected for generation of a conditionally activebiologic protein. Certain non-limiting examples will be discussed infurther detail below.

Therapeutic proteins are those which can be used in medicine eitheralone or in conjunction with other therapies to treat various diseasesor medical conditions, such as antibodies, enzymes, immune regulators,growth factors, hormones and cytokines. The conditionally activebiologic proteins of the disclosure could be appropriate for use in oneor more indications including the treatment of circulatory disorders,arthritis, multiple sclerosis, autoimmune disorders, cancer,dermatologic conditions and use in various diagnostic formats. Dependingon the protein and indication, the conditionally active biologic proteincould be administered in parenteral, topical or oral formulations asdiscussed below.

Some representative target wild-type proteins include enzymes,antibodies, cytokines, receptors, DNA binding proteins, chelatingagents, and hormones. More examples include industrial andpharmaceutical proteins, such as ligands, cell surface receptors,antigens, transcription factors, signaling modules, and cytoskeletalproteins. Some suitable classes of enzymes are hydrolases such asproteases, carbohydrases, lipases; isomerases such as racemases,epimerases, tautomerases, or mutases; transferases, kinases,oxidoreductases, and phophatases.

The target wild-type proteins can be discovered by generating andscreening a library for a protein with a desired properties, such asenzymatic activity, binding affinity/selectivity, thermostability,tolerance of high or low pH, expression efficiency, or other biologicalactivities.

The target wild-type proteins may be discovered by screening a cDNAlibrary. A cDNA library is a combination of cloned cDNA (complementaryDNA) fragments inserted into a collection of host cells, which togetherconstitute some portion of the transcriptome of the organism. cDNA isproduced from fully transcribed mRNA and therefore contains the codingsequence for expressed proteins of an organism. The information in cDNAlibraries is a powerful and useful tool for discovery of proteins withdesired properties by screening the libraries for proteins with thedesire property.

In embodiments where the target wild-type proteins are antibodies, thewild-type antibodies can be discovered by generating and screeningantibody libraries. The antibody libraries can be either polyclonalantibody libraries or monoclonal antibody libraries. A polyclonalantibody library against an antigen can be generated by direct injectionof the antigen into an animal or by administering the antigen to anon-human animal. The antibodies so obtained represent a library ofpolyclonal antibodies that bind to the antigen. For preparation ofmonoclonal antibody libraries, any technique which provides antibodiesproduced by continuous cell line cultures can be used. Examples includethe hybridoma technique, the trioma technique, the human B-cellhybridoma technique, and the EBV-hybridoma technique (see, e.g., Cole(1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96). Techniques described for the generating single chainantibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted toproduce a single chain antibody library.

There are other methods for generation and screening of antibodylibraries for discovery of the wild-type antibody. For example, fullyhuman antibody display libraries can be utilized. Such a library is apopulation of antibodies displayed on the surface of host cell(s).Preferably, the antibody library is representative of the humanrepertoire of antibodies in that they have the capability of binding toa wide range of antigens. Because the antibodies are displayed on thesurface of cells, the effective affinity (due to avidity) of eachantibody in the library is increased. Unlike other popular librarytypes, such as phage display libraries, where avidity of the antibodiesfor screening and identification purposes is less desirable, the superavidity provided by cell surface display in the present invention, isdesirable. Cell surface display libraries enable the identification oflow, medium and high binding affinity antibodies, as well as theidentification of non-immunogenic and weak epitopes in the screening orselection step.

Circulatory Disorders-Thrombosis and Thrombolytic Therapy.

A thrombus (blood clot) is defined as a solid mass derived from bloodconstituents that forms in the circulatory system. The thrombus isformed by a series of events involving blood coagulation factors,platelets, red blood cells, and interactions with the vessel wall. Aplatelet is an intravascular aggregation of platelets, fibrin andentrapped blood cells which can cause vascular obstruction. Byobstructing or blocking blood flow, the thrombus deprives downstreamtissue of oxygen supply. Fragments (emboli) of the thrombus may breakaway and obstruct smaller vessels. Arterial thrombus formation isprecipitated by any of a variety of factors including an underlyingstenosis-atherosclerosis, a low flow state-cardiac function,hypercoagubility as in cancer or a coagulation factor deficiency, or aforeign body such as a stent or catheter. A thrombus leading to arterialischemia can result in limb or tissue injury, acute myocardialinfarction (AMI), stroke, amputation, or bowel infarction. Major causesof morbidity and mortality are the formation of arterial thrombi(coronary arterial thrombi and cerebral arterial thrombi) and pulmonarythrombi. Venous thrombus formation can occur due to endothelial injurysuch as trauma, stasis due to e.g. immobility, or hypercoagulability,but atherosclerosos is not a factor. Treatment strategies includemechanical thrombectomy, pharmacomechanical thrombectomy andthrombolysis. Thrombotic therapy is used to minimize formation and aidin removal of thrombi.

Thrombotic therapy includes the use of antiplatelet agents which inhibitplatelet activation, anticoagulant therapies, and/or thrombolytictherapy to degrade blood clots. Examples of antiplatelets includeaspirin, dipyridamole, and ticlopidine. Examples of anticoagulantsinclude heparin, warfarin, hirudin, and activated human protein C.Examples of thrombolytics include tissue plasminogen activator (tPA)/tPAvariants, urokinase and streptokinase. The thrombolytics display acatalytic mode of action.

Thrombolytic therapy in acute myocardial infarction is well established.Use of thrombolytic agents has become standard emergency treatment.Although effective, these products achieve complete reperfusion in onlyabout 50% of patients and side effects include risk of hemorrhage (inparticular intracranial bleeding) as well as hypertension. Thedegradation of blood clots from a damaged or diseased vessel is termed“fibrinolysis” or the “fibrinolytic process”. Fibrinolysis is aproteolytic process, by a plasminogen activator which activates theprotein plasminogen, thereby forming plasmin. Plasmin proteolyticallydegrades the fibrin strands of the blood clot to dissolve the clot.Fibrin specific plasminogen activators include tissue plasminogenactivators or variants. Non-specific plasminogen activators can includestreptokinase and urokinase.

Certain commonly used thrombolytic therapies utilize one of severalavailable tissue plasminogen activator (tPA) variants. For example, tPAbased product variants which have been previously approved for use areAlteplase (rt-PA), Reteplase (r-PA) and Tenecteplase (TNK). Approveduses for tPA variants include, for example, acute myocardial infarctionfor the improvement of ventricular function following AMI, the reductionof incidence of congestive heart failure, and reduction of mortalityassociated with AMI, management of ischemic stroke in adults forimproving neurological recovery and reducing incidence of disability,management of acute massive pulmonary embolism in adults for the lysisof acute pulmonary emboli, and for the lysis of pulmonary emboliaccompanied by unstable hemodynamics.

Another commonly used thrombolytic therapy utilizes urokinase. Urokinaseis a standard lytic agent used in the management of peripheral vasculardisease.

Streptokinase is a protein secreted by several species of streptococcithat can bind and activate human plasminogen. Complexes of streptokinasewith human plasminogen can hydrolytically activate other unboundplasminogen by activating through bond cleavage to produce plasmin. Theusual activation of plasminogen is through the proteolysis of theArg561-Val562 bond. The amino group of Val562 then forms a salt-bridgewith Asp740, which causes a conformational change to produce the activeprotease plasmin. Plasmin is produced in the blood to break down fibrin,the major constituent of blood clots.

Streptokinase is used as an effective clot-dissolving medication in somecases of myocardial infarction (heart attack), pulmonary embolism (lungblood clots), and deep venous thrombosis (leg blood clots).Streptokinase belongs to a group of medications called fibrinolytics.Streptokinase is given as soon as possible after the onset of a heartattack to dissolve clots in the arteries of the heart wall and reducedamage to the heart muscle. Streptokinase is a bacterial product, so thebody has the ability to build up immunity against the protein.Therefore, it is recommended that this product should not be given againafter four days from the first administration, as it may not be aseffective and cause an allergic reaction. For this reason it is usuallygiven only after a first heart attack, and further thrombotic events aretypically treated with tissue plasminogen activator (TPA). Streptokinaseis also sometimes used to prevent post-operative adhesions.

Side effects of streptokinase include bleeding (major and minor),hypotension, and respiratory depression as well as possible allergicreaction. In addition, anticoagulants, agents that alter plateletfunction (e.g. aspirin, other NSAIDs, dipyridamole) may increase risk ofbleeding.

Administration of the thrombolytics is generally by infusion or by bolusintravenous dose; or by a mechanical infusion system. Adverse effectscan include serious intracranial, gastrointestinal, retroperitoneal, orpericardial bleeding. If bleeding occurs the administration must bediscontinued immediately.

In certain embodiments of the disclosure, tPA, streptokinase orurokinase is selected as the target, or wild-type protein.

In one embodiment, the methods of the disclosure are used to select fora conditionally active recombinant or synthetic streptokinase variantwith high activity at aberrant temperature conditions below normalphysiological conditions; and substantial deactivation or inactivationat normal physiological conditions (e.g. 37 degrees C.). In one aspect,the aberrant temperature condition is room temperature, e.g. 20-25degrees C. In another aspect, the disclosure provides a method oftreating a stroke or heart attack, the method comprising administering ahigh dose of the conditionally active streptokinase variant to stroke orheart attack victims in order to clear clots, yet allow for rapidinactivation of the streptokinase variant to avoid excessive bleeding.

Circulatory Disorders-Renin/Angiotensin

The renin-angiotensin system is a hormone system that regulates bloodpressure and water (fluid) balance. The kidneys secrete renin when theblood volume is low. Renin is an enzyme which hydrolyzes angiotensinogensecreted from the liver into the peptide angiotensin I. Angiotensin I isfurther cleaved in the lungs by endothelial-bound angiotensin convertingenzyme (ACE) into angiotensin II, the most vasoactive peptide.Angiotensin II causes the blood vessels to constrict, resulting inincreased blood pressure. However, angiotensin π also stimulates thesecretion of the hormone aldosterone from the adrenal cortex.Aldosterone causes the tubules of the kidneys to increase the resorptionof sodium and water. This increases the volume of fluid in the body,which also increases blood pressure. An over-active renin-angiotensinsystem leads to vasoconstriction and retention of sodium and water.These effects lead to hypertension. There are many drugs which interruptdifferent steps in this system to lower blood pressure. These drugs areone of the main ways to control high blood pressure (hypertension),heart failure, kidney failure, and harmful effects of diabetes.

Hypovolemic shock is an emergency condition in which severe blood and/orfluid loss makes the heart unable to adequately perfuse the body's cellswith oxygenated blood. Blood loss can be from trauma, injuries andinternal bleeding. The amount of circulating blood may drop due toexcessive fluid loss from burns, diarrhea, excessive perspiration orvomiting. Symptoms of hypovolemic shock include anxiety, cool clammyskin, confusion, rapid breathing, or unconsciousness. Examination showssigns of shock including low blood pressure, low body temperature, andrapid pulse, which may be weak or thready. Treatment includesintravenous fluids; blood or blood products; treatment for shock; andmedication such as dopamine, dobutamine, epinephrine and norepinephrineto increase blood pressure and cardiac output.

In one embodiment, the disclosure provides a method of selecting for aconditionally active recombinant renin variant to be reversiblydeactivated at normal physiological temperature, but reactivated at theaberrant lower temperatures in a patient with hypovolemic shock. Theconditionally active protein can be used to treat hypovolemic shock tohelp increase the volume of fluid in the body, and increase bloodpressure. Circulatory Disorders-Reynaud's phenomenon

Reynaud's phenomenon (RP) is a vasospastic disorder causingdiscoloration of the fingers, toes and occasionally other extremities.Emotional stress and cold are classic triggers of the phenomenon. Whenexposed to cold temperatures, the extremities lose heat. The bloodsupply to fingers and toes is normally slowed to preserve the body'score temperature. Blood flow is reduced by the narrowing of smallarteries under the skin of the extremities. Stress causes similarreaction to cold in the body. Li Reynaud's, the normal response isexaggerated. The condition can cause pain, discoloration, and sensationsof cold and numbness. The phenomenon is the result of vasospasms thatdecrease the blood supply to the respective regions, in Reynaud'sdisease (Primary Raynaud's phenomenon), the disease is idiopathic. LiRaynaud's syndrome (Secondary Reynaud's), the phenomenon is caused bysome other instigating factor. Measurement of hand-temperature gradientsis one tool to distinguish between the primary and secondary forms. Theprimary form can progress to the secondary form, and in extreme cases,the secondary form can progress to necrosis or gangrene of thefingertips.

Raynaud's phenomenon is an exaggeration of responses to cold oremotional stress. Primary RP is essentially mediated by microvascularvasospasm. Hyperactivation of the sympathetic system causes extremevasoconstriction of the peripheral blood vessels, leading to hypoxia.Chronic, recurrent cases can result in atrophy of the skin, subcutaneoustissue, and muscle. It can also rarely result in ulceration and ischemicgangrene.

Traditional treatment options for Reynaud's phenomenon includeprescription medication that dilates blood vessels and promotescirculation. These include calcium channel blockers, such as nifedipineor diltiazem; alpha blockers, which counteract the actions ofnorepinephrine, a hormone that constricts blood vessels, such asprazosin or doxazosin; and vasodilators, to relax blood vessels, such asnitroglycerin cream, or the angiotensin II inhibitor losartan,sildenafil, or prostaglandins. Fluoxetine, a selective serotoninreuptake inhibitor and other antidepressant medications may reduce thefrequency and severity of episodes due to psychological stressors. Thesedrugs may cause side effects such as headache, flushing and ankle edema.A drug may also lose effectiveness over time.

The regulation of cutaneous vasoconstriction and vasodilation involvesaltered sympathetic nerve activity and a number of neuronal regulators,including adrenergic and non-adrenergic, as well as REDOX signaling andother signaling such as the RhoA/ROCK pathway. Vasoconstriction ofvascular smooth muscle cells (vSMC) in the skin is thought to beactivated by norepinephrine mediated by alpha1 and alpha2adrenoreceptors. Alpha2C-ARs translocate from the trans Golgi to thecell surface of the vSMC where they respond to stimulation and signalingof these responses involves the RhoA/Rhokinase (ROCK) signaling pathway.Cold stimulation in cutaneous arteries results in the immediategeneration of reactive oxygen species (ROS) in the vSMC mitochondria.ROS are involved in the REDOX signaling through the RhoA/ROCK pathway.RhoA is a GTP-binding protein whose role is the regulation ofactin-myosin dependent processes such as migration and cell contractionin vSMC. Non-adrenergic neuropeptides with known function in vasculaturewith possible involvement in RP include calcitonin gene-related peptide(CGRP), Substance P (SP), Neuropeptide Y (NPY), and vasoactiveintestinal peptide (VIP). Fonseca et al., 2009, “Neuronal regulators andvascular dysfunction in Raynaud's phenomenon and systemic sclerosis”,Curr. Vascul. Pharmacol. 7:34-39.

New therapies for RP include alpha-2c adrenergic receptor blockers,protein tyrosine kinase inhibitors, Rho-kinase inhibitors and calcitoningene related peptide.

Calcitonin gene related peptide (CGRP) is a member of the calcitoninfamily of peptides and exists in two forms; alpha-CGRP and beta-CGRP.Alpha-CGRP is a 37-amino acid peptide formed from alternative splicingof the calcitonin/CGRP gene. CGRP is one of the most abundant peptidesproduced in peripheral and central neurons. It is a potent peptidevasodilator and can function in the transmission of pain. Migraine is acommon neurological disorder that is associated with an increase in CGRPlevels. CGRP dilates intracranial blood vessels and transmits vascularnociception. CGRP receptor antagonists have been tested as treatmentsfor migraines. Arulmani et al., 2004, “Calcitonin gene-related peptideand it role in migraine pathophysiology”, Eur. J. Pharmacol. 500(1-3):315-330. At least three receptor subtypes have been identified and CGRPacts through G protein-coupled receptors whose presence and changes infunction modulate the peptide's effect in various tissues. CGRP's signaltransduction through the receptors is dependent on two accessoryproteins: receptor activity modifying protein 1 (RAMP1) and receptorcomponent protein (RCP). Ghatta 2004, Calcitonin gene-related peptide:understanding its role. Indian J. Pharmacol. 36(5): 277-283. One studyof the effects of intravenous infusion of three vasodilators:endothelium-dependent vasodilator adenosine triphosphate (ATP),endothelium-independent vasodilator prostacyclin (epoprostenol; PGI2),and CGRP, to patients with Reynaud's phenomenon, and a similar number ofage and sex matched controls, using laser Doppler flowmetry (LDF) showedCGRP induced flushing of the face and hands by a rise in skin blood flowin the Reynaud's patients, whereas in controls CGRP caused flushing onlyin the face. PGI2 caused similar rises in blood flow in hands and faceof both groups. ATP did not cause any significant changes in blood flowin hands or face of the patients, but increased blood flow to the faceof controls. Shawket et al., 1989, “Selective suprasensitivity tocalcitonin-gene-related peptide in the hands in Reynaud's phenomenon”.The Lancet, 334(8676):1354-1357. In one aspect, the wild-type proteintarget molecule is CGRP.

In one embodiment, the disclosure provides methods of selecting forconditionally active recombinant protein variants of proteins associatedwith Reynaud's syndrome to be reversibly deactivated at normalphysiological temperature, but reactivated at the aberrant lowertemperatures in digits. The conditionally active proteins can be used totreat Reynaud's phenomenon, to prevent or reduce loss of digit functiondue to low circulation. Circulatory disorders-Vasopressin

Arginine vasopressin (AVP, vasopressin, antidiuretic hormone (ADH)) is apeptide hormone found in most mammals that controls reabsorption ofmolecules in the tubules of the kidney by affecting tissue permeability.One of the most important roles of vasopressin is to regulate waterretention in the body. In high concentrations it raises blood pressureby introducing moderate vasoconstriction. Vasopressin has three effectswhich result in increased urine osmolality (increased concentration) anddecreased water excretion. First, vasopressin causes an increase in thepermeability of water of the collecting duct cells in the kidneyallowing water resorption and excretion of a smaller volume ofconcentrated urine (antidiuresis). This occurs through insertion ofaquaporin-2 water channels into the apical membrane of the collectingduct cells. Secondly, vasopressin causes an increase in the permeabilityof the inner medullary portion of the collecting duct to urea, allowingincreased reabsorption urea into the medullary interstitium. Thirdly,vasopressin causes stimulation of sodium and chloride reabsorption inthe thick ascending limb of the loop of Heme by increasing the activityof the Na⁺-K⁺-2Cl^(″)-cotransporter. NaCl reabsorption drives theprocess of countercurrent multiplication, which furnishes the osmoticgradient for aquaporin mediated water reabsorption in the medullarycollecting ducts.

The hypertonic interstitial fluid surrounding the collecting ducts ofthe kidney provides a high osmotic pressure for the removal of water.Transmembrane channels made of proteins called aquaporins are insertedin the plasma membrane greatly increasing its permeability to water.When open, an aquaporin channel allows 3 billion molecules of water topass through each second. Insertion of aquaporin-2 channels requiressignaling by vasopressin. Vasopressin binds to receptors (called V2receptors) on the basolateral surface of the cells of the collectingducts. Binding of the hormone triggers a rising level of cAMP within thecell. This “second messenger” initiates a chain of events culminating inthe insertion of aquaporin-2 channels in the apical surface of thecollecting duct cells. The aquaporins allow water to move out of thenephron, increasing the amount of water re-absorbed from the formingurine back into the bloodstream.

The main stimulus for the release of vasopressin from the pituitarygland is increased osmolality of the blood plasma. Anything thatdehydrates the body, such as perspiring heavily increases the osmoticpressure of the blood and turns on the vasopressin to V2 receptor toaquaporin-2 pathway. As a result, as little as 0.5 liters/day of urinemay remain of the original 180 liters/day of nephric filtrate. Theconcentration of salts in urine can be as high as four times that of theblood. If the blood should become too dilute, as would occur fromdrinking a large amount of water, vasopressin secretion is inhibited andthe aquaporin-2 channels are taken back into the cell by endocytosis.The result is that a large volume of watery urine is formed with a saltconcentration as little as one-fourth of that of the blood.

Decreased vasopressin release or decreased renal sensitivity to AVPleads to diabetes insipidus, a condition featuring hypernatremia(increased blood sodium concentration), polyuria (excess urineproduction), and polydipsia (thirst).

High levels of AVP secretion (syndrome of inappropriate antidiuretichormone, SIADH) and resultant hyponatremia (low blood sodium levels)occurs in brain diseases and conditions of the lungs (Small cell lungcarcinoma). In the perioperative period, the effects of surgical stressand some commonly used medications (e.g., opiates, syntocinon,anti-emetics) lead to a similar state of excess vasopressin secretion.This may cause mild hyponatremia for several days.

Vasopressin agonists are used therapeutically in various conditions, andits long-acting synthetic analogue desmopressin is used in conditionsfeaturing low vasopressin secretion, as well as for control of bleeding(in some forms of von Willebrand disease) and in extreme cases ofbedwetting by children. Terlipressin and related analogues are used asvasoconstrictors in certain conditions. Vasopressin infusion has beenused as a second line of management in septic shock patients notresponding to high dose of inotropes (e.g., dopamine or norepinephrine).A vasopressin receptor antagonist is an agent that interferes withaction at the vasopressin receptors. They can be used in the treatmentof hyponatremia.

In one embodiment, the disclosure provides methods to select forconditionally active biologic recombinant or synthetic protein variantsof proteins involved in the vasopressin response to be reversiblydeactivated at normal physiological osmotic pressure, but reactivated ataberrant osmotic pressure in the blood. In another embodiment, variantsof proteins involved in the vasopressin response are activated underhyponatremic conditions, but inactivated at normal serum sodiumconcentrations. In one aspect, hyponatremic conditions are those whereserum sodium<135 mEq/L.

Cancer-Angiostatin

Angiostatin is a naturally occurring protein in several animal species.It acts as an endogenous angiogenesis inhibitor (i.e., it blocks thegrowth of new blood vessels). Angiostatin is able to suppress tumor cellgrowth and metastasis through inhibition of endothelial cellproliferation and migration. Angiostatin is a 38 kD fragment of plasmin(which is itself a fragment of plasminogen). Angiostatin comprises thekringles 1 to 3 of plasminogen. Angiostatin is produced, for example, byautolytic cleavage of plasminogen, involving extracellular disulfidebond reduction by phosphoglycerate kinase. Angiostatin can also becleaved from plasminogen by different matrix metalloproteinases (MMPs)including MMP2, MMP 12 and MMP9, and serine proteases (neutrophilelastase, prostate-specific antigen (PSA)). In vivo angiostatin inhibitstumor growth and keeps experimental metastasis in a dormant state.Angiostatin is elevated in animals with primary tumors and otherinflammatory and degenerative diseases.

Angiostatin is known to bind many proteins including angiomotin andendothelial cell surface ATO synthase, but also integrins, annexin II,C-met receptor, NG2-proteoglycans, tissue-plasminogen activator,chondroitin sulfate glycoproteins, and CD26. One study shows that IL-12,a TH1 cytokine with potent antiangiogenic activity, is a mediator ofangiostatin's activity. Albin”, J. Translational Medicine. Jan. 4, 2009,7:5. Angiostatin binds and inhibits ATP synthase on the endothelial cellsurface. ATP synthase also occurs on the surface of a variety of cancercells. Tumor cell surface ATP synthase was found to be more active atlow extracellular pH; a hallmark of tumor microenvironment. Angiostatinwas found to affect tumor cell surface ATP synthase activity at acidicextracellular pH (pHe). At low extracellular pH, angiostatin wasdirectly anti-tumorigenic. At low pH, angiostatin and anti-beta-subunitantibody induce intracellular acidification of A549 cancer cells, aswell as a direct toxicity that is absent in tumor cells with low levelsof extracellular ATP synthase. It was hypothesized that the mechanism oftumor cytotoxicity is dependent on intracellular pH deregulation due toinhibition of cell surface ATP synthase. Chi and Pizzo, “Angiostatin isdirectly cytotoxic to tumor cells at low extracellular pH: a mechanismdependent on cell surface-associated ATP synthase”, Cancer Res., 2006,66(2): 875-82.

In one embodiment, the disclosure provides a method for identificationof conditionally active angiostatin variant which is less active thanwild-type angiostatin at normal physiological blood pH, but exhibitsenhanced activity at low pH. Low pH is defined as being less than normalphysiological pH. In one aspect, low pH is less than about pH 7.2. In aparticular aspect, low pH is about pH 6.7.

In one aspect, the conditionally active angiostatin variant can beformulated and utilized as an anticancer agent.

Autoimmune Disease-Conditionally Active Biological Response Modifiers

Rheumatoid arthritis is an autoimmune disease characterized by aberrantimmune mechanisms that lead to joint inflammation and swelling withprogressive destruction of the joints. RA can also affect the skin,connective tissue and organs in the body. Traditional treatment includesnon-steroidal anti-inflammatory drugs (NSAIDS), COX-2 inhibitors, anddisease-modifying anti-rheumatic drugs (DMARDS) such as methotrexate.None of the traditional treatment regimes is ideal, especially for longterm use.

Biological response modifiers, which target inflammatory mediators,offer a relatively new approach to the treatment of rheumatoid arthritisand other autoimmune diseases. Such biological response modifiersinclude antibodies, or active portions thereof, against variousinflammatory mediators such as IL-6, IL-6 receptor, TNF-alpha, IL-23 andIL-12.

Some of the first biological response modifiers were medicationstargeting tumor necrosis factor alpha (TNF-a), a pro-inflammatorycytokine involved in the pathogenesis of RA. Several anti-TNF-alphamedications are currently marketed for the treatment of RA. For example,Enbrel® (etanercept, Amgen) is a TNF-alpha blocker. Etanercept is adimeric fusion protein consisting of the extracellular ligand-bindingportion of the human 75 kilodalton (p75) tumor necrosis factor receptor(TNFR) linked to the Fc portion of human IgG1. The Fc component ofetanercept contains the CH2 domain, the CH3 domain and hinge region, butnot the CH1 domain of IgG1. Etanercept is produced in a Chinese hamsterovary (CHO) mammalian cell expression system. It consists of 934 aminoacids and an apparent molecular weight of about 150 kilodaltons. Enbrel®is used to treat rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis and plaque psoriasis. Serious side effects of Enbrel®include infections including tuberculosis, fungal infection, bacterialor viral infection due to opportunistic pathogens. Sepsis can alsooccur. Lymphoma, or other malignancies have also been reported.

Remicade® (infliximab) is a chimeric anti-TNF-alpha IgGkI monoclonalantibody composed of human constant and murine variable regions.Remicade is administered by intravenous injection and is used to treatrheumatoid arthritis, psoriasis, Crohn's disease, ulcerative colitis,and ankylosing spondylitis. Side effects of Remicade include seriousinfection or sepsis, and rarely certain T-cell lymphomas. Other sideeffects include hepatotoxicity, certain severe hematologic events,hypersensitivity reactions and certain severe neurological events.

Other biological response modifiers include humanized anti-interleukin-6(IL-6) receptor antibodies. IL-6 is a cytokine that contributes toinflammation, swelling and joint damage in RA. One humanized anti-IL-6receptor antibody, Actemra (tocilizumab, Roche), is approved by the FDAand European Commission to treat adult patients with rheumatoidarthritis. Actemra is also approved in Japan for treatment of RA andjuvenile idiopathic arthritis (sJIA). Phase III studies showed thattreatment with Actemra as a monotherapy, or a combination with MTX orother DMARDs, reduced signs and symptoms of RA compared with othertherapies. Actemra is a humanized anti-human IL-6 receptor monoclonalantibody that competitively blocks the binding of IL-6 to its receptor.Thus, it inhibits the proliferative effects of IL-6, which lead tosynovial thickening and pannus formation in RA. Serious side effects ofActemra, include serious infections and hypersensitivity reactionsincluding a few cases of anaphylaxis. Other side effects include upperrespiratory tract infection, headache, nasopharyngitis, hypertension andincreased ALT.

Another common autoimmune disease is psoriasis. An overactive immunesystem can lead to high levels of IL-12 and IL-23, two cytokine proteinsthat have been found in psoriatic skin plaques. IL-12 and IL-23 areinvolved in inflammatory and immune responses such as natural killercell activation and CD4+ T-cell differentiation and activation.

One treatment for moderate or severe psoriasis involves subcutaneousinjection of Stelara™ (ustekinumab, Centocor Ortho Biotech, Inc.) ahumanized IgGIk monoclonal antibody against the p40 subunit of the IL-12and IL-23 cytokines. Stelara has been shown to provide relief fromcertain symptoms associated with psoriatic plaques, such as plaquethickness, scaling and redness. The formulation for Stelara includesL-histidine and L-histidine monohydrochloride monohydrate, polysorbate80, and sucrose in aqueous solution. Use of Stelara™ affects the immunesystem, and may increase chances of infection, including tuberculosis,and infections caused by bacteria, fungi or viruses; as well as increasethe risk of certain types of cancer.

Side effects of the biological response modifiers are significant andare caused in part by high levels following injection into patientsrenders patients susceptible to serious infection or death. This is amajor side effect associated with this important class of drugs. Onechallenge is avoiding the high initial level of activity from the doseof antibody required to provide a long treatment effect followinginjection.

Conditionally Active Biological Antibodies for Brains

It has long been a challenge to deliver drugs, especially largemolecules such as antibodies, to the brain because brain penetration bydrugs is severely limited by the largely impermeable BBB. Fortunately,the BBB has endogenous transport systems that are mediated by a BBBreceptor (BBB-R), which is a specific receptor that allows transport ofmacromolecules across the BBB. For example, an antibody that can bind toa BBB-R may be transported across BBB using the endogenous transportsystems. Such an antibody may serve as a vehicle for transport of drugsor other agents across BBB by using the endogenous BBB receptor mediatedtransport system that traverses the BBB. Such antibodies need not havehigh affinity to a BBB-R. Antibodies that are not conditionally activeantibodies with low affinities for BBB-R have been described as crossingthe BBB more efficiently than a high affinity antibody, as described inUS 2012/0171120 (incorporated herein by reference). Unlike traditionalantibodies, conditionally active antibodies are not required to have lowaffinity for BBB-R to cross the BBB and remain inside the brain.Conditionally active antibodies can have high affinity for the BBB-R onthe blood side of the BBB, and little or no affinity on the brain sideof the BBB. Drugs, such as drug conjugates, may be coupled to aconditionally active antibody to be transported with the antibody acrossthe BBB into the brain.

A BBB-R is a transmembrane receptor protein expressed on brainendothelial cells which is capable of transporting molecules across theblood-brain barrier. Examples of BBB-R include transferrin receptor(TfR), insulin receptor, insulin-like growth factor receptor (IGF-R),low density lipoprotein receptors including without limitation lowdensity lipoprotein receptor-related protein 1 (LRP1) and low densitylipoprotein receptor-related protein 8 (LRP8), and heparin-bindingepidermal growth factor-like growth factor (HB-EGF). An exemplary BBB-Rherein is a transferrin receptor (TfR). The TfR is a transmembraneglycoprotein (with a molecular weight of about 180,000) composed of twodisulphide-bonded sub-units (each of apparent molecular weight of about90,000) involved in iron uptake in vertebrates.

In some embodiments, the present invention provides a conditionallyactive antibody generated from a parent or wild-type antibody against aBBB-R. The conditionally active antibody binds the BBB-R on the bloodside of the BBB, and has a lower affinity to the BBB-R than the parentor wild-type antibody on the brain side of the BBB. In some otherembodiments, the conditionally active antibody has affinity to the BBB-Rthan the wild type or parent antibody on the blood side of the BBB, andhas no affinity to the BBB-R on the brain side of the BBB.

Blood plasma is a body fluid that is very different from brainextracellular fluid (ECF). As discussed by Somjen (“Ions in the Brain:Normal Function, Seizures, and Stroke,” Oxford University Press, 2004,pages 16 and 33) and Redzic (“Molecular biology of the blood-brain andthe blood-cerebrospinal fluid barriers: similarities and differences,”Fluids and Barriers of the CNS, vol. 8:3, 2011), the brain extracellularfluid has significantly less K⁺, more Mg²⁺ and H⁺ than blood plasma. Thedifferences in ion concentrations between blood plasma and brain ECFlead to significant differences in osmotic pressure and osmolalitybetween the two fluids. Table 1 shows the concentrations of common ionsin millimoles for both blood plasma and brain ECF.

TABLE 1 Common ions in plasma (arterial plasma) and brain extracellularfluid (CSF) ARTERIAL PLASMA CSF HUMAN RAT HUMAN RAT Na⁺ 150 148 147 152K⁺ 4.6 5.3 2.9 3.4 Ca, total 2.4 3.1 1.14 1.1 Ca²⁺, free 1.4 1.5 1.0 1.0pCa Mg, total 0.86 0.8 1.15 1.3 Mg²⁺, free 0.47 0.44 0.7 0.88 H⁺0.000039 0.000032 0.000047 0.00005 pH 7.41 7.5 7.3 7.3 Cl⁻ 99 119 HCO₃ ⁻26.8 31 23.3 28

Brain ECF also contains significantly more lactate than blood plasma andsignificantly less glucose than blood plasma (Abi-Saab et al., “StrikingDifferences in Glucose and Lactate Levels Between Brain ExtracellularFluid and Plasma in Conscious Human Subjects: Effects of Hyperglycemiaand Hypoglycemia,” Journal of Cerebral Blood Flow & Metabolism, vol. 22,pages 271-279, 2002).

Thus, there are several physiological conditions that are differentbetween the two sides of the BBB, such as pH, concentrations of varioussubstances (such as lactose, glucose, K+, Mg2+), osmotic pressure andosmolality. For the physiological condition of pH, human blood plasmahas a higher pH than human brain ECF. For the physiological condition ofK+concentration , brain ECF has a lower K+ concentration than humanblood plasma. For the physiological condition of Mg2+ concentration, thehuman brain ECF has significantly more Mg2+ than human blood plasma. Forthe physiological condition of osmotic pressure, the human brain ECF hasan osmotic pressure that is different from that of human blood plasma.In some embodiments, the physiological conditions of brain ECF may bethe composition, pH, osmotic pressure and osmolality of brain ECF ofpatients with a particular neurological disorder, which may be differentfrom the physiological condition of the brain ECF of the generalpopulation.

The present invention thus provides a method for evolving a DNA thatencodes a template antibody against a BBB-R to create a mutant DNAlibrary. The mutant DNA library is then expressed to obtain mutantantibodies. The mutant antibodies are screened for a conditionallyactive antibody that has binds to the BBB-R under at least one bloodplasma physiological condition and has a low or no affinity to the BBB-Runder at least one brain physiological condition in the brain ECFcompared to the template antibody. Thus, the selected mutant antibodyhas a low or high affinity to the BBB-R at the blood plasma side and alow or no affinity to the BBB-R at the brain ECF side. This selectedmutant antibody is useful as a conditionally active antibody fortransport across the BBB.

Such a conditionally active antibody is advantageous for crossing theBBB and remaining in the brain ECF. The low affinity to the BBB-R at thebrain side lowers the rate (or removes) the conditionally activeantibody is transported back across the BBB out of the brain and backinto the blood relative to the template antibody.

In some other embodiments, the present invention provides a method forevolving a DNA that encodes a template antibody against a BBB-R tocreate a mutant DNA library. The mutant DNA library is then expressed toobtain mutant antibodies. The mutant antibodies are screened for aconditionally active antibody that binds to the BBB-R under at least oneblood plasma physiological condition and little or no affinity to theBBB-R under at least one brain physiological condition. Thus, theselected mutant antibody has affinity to the BBB-R at the plasma sideand little or no affinity to the BBB-R at the brain ECF side. Thisselected mutant antibody is a conditionally active antibody.

Such a conditionally active antibody is advantageous in crossing the BBBand remaining in the brain ECF. After binding to the BBB-R at the bloodplasma side, the conditionally active antibody is transported across theBBB, and the little to no affinity to the BBB-R at the brain ECF sidemeans that the conditionally active antibody is unlikely to betransported out of the brain.

The affinity of the conditionally active antibody to a BBB-R may bemeasured by its half maximal inhibitory concentration (IC50), which is ameasure of how much of the antibody is needed to inhibit the binding ofa known BBB-R ligand to the BBB-R by 50%. A common approach is toperform a competitive binding assay, such as competitive ELISA assay. Anexemplary competitive ELISA assay to measure IC50 on TfR (a BBB-R) isone in which increasing concentrations of anti-TfR antibody competeagainst biotinylated TfR^(A) for binding to TfR. The anti-TfR antibodycompetitive ELISA may be performed in Maxisorp plates (Neptune, N.J.)coated with 2.5 μg/ml of purified murine TfR extracellular domain in PBSat 4° C. overnight. Plates are washed with PBS/0.05% Tween 20 andblocked using Superblock blocking buffer in PBS (Thermo Scientific,Hudson, N.H.). A titration of each individual anti-TfR antibody (1:3serial dilution) is combined with biotinylated anti-TfR^(A) (0.5 nMfinal concentration) and added to the plate for 1 hour at roomtemperature. Plates are washed with PBS/0.05% Tween 20, andHRP-streptavidin (Southern Biotech, Birmingham) is added to the plateand incubated for 1 hour at room temperature. Plates are washed withPBS/0.05% Tween 20, and biotinylated anti-TfR^(A) bound to the plate isdetected using TMB substrate (BioFX Laboratories, Owings Mills).

A high IC50 indicates that more of the conditionally active antibody isrequired to inhibit binding of the known ligand of a BBB-R, and thusthat the antibody's affinity for that BBB-R is relatively low.Conversely, a low IC50 indicates that less of the conditionally activeantibody is required to inhibit binding of the known ligand, and thusthat the antibody's affinity for that BBB-R is relatively high.

In some embodiments, the IC50 of the conditionally active antibodiesfrom a BBB-R in the blood plasma may be from about 1 nM to about 100 μM,or from about 5 nM to about 100 μM, or from about 50 nM to about 100 μM,or from about 100 nM to about 100 μM, or from about 5 nM to about 10 μM,or from about 30 nM to about 1 μM, or from about 50 nM to about 1 μM.

Conditionally Active Biological Proteins for Synovial Fluid

Joint diseases are a major cause of disability and early retirement inthe industrialized countries. Joint diseases often lead to damage at ajoint which is difficult to repair. Synovial fluid is a body fluid thatis found in the synovial cavity of the joints (e.g., knee, hip,shoulder) of a human or animal body between the cartilage and synoviumof facing articulating surfaces. Synovial fluid provides nourishment tothe cartilage and also serves as a lubricant for the joints. The cellsof the cartilage and synovium secrete fluid that serve as a lubricantbetween the articulating surfaces. Human synovial fluid comprisesapproximately 85% water. It is derived from the dialysate of bloodplasma, which itself is made up of water, dissolved proteins, glucose,clotting factors, mineral ions, hormones, etc. Proteins such as albuminand globulins are present in synovial fluid and are believed to play animportant role in the lubricating the joint area. Some other proteinsare also found in human synovial fluid, including the glycoproteins suchas alpha-1-acid glycoprotein (AGP), alpha-1-antitrypsin (A1AT) andlubricin.

Synovial fluid has a composition that is very different from other partsof the body. Thus, synovial fluid has physiological conditions that aredifferent from other parts of the body, such as the blood plasma. Forexample, synovial fluid has less than about 10 mg/dL of glucose whereasthe mean normal glucose level in human blood plasma is about 100 mg/dL,fluctuating within a range between 70 and 100 mg/dL throughout the day.In addition, the total protein level in the synovial fluid is about onethird of the blood plasma protein level since large molecules such asproteins do not easily pass through the synovial membrane into thesynovial fluid. It has also been found that the pH of human synovialfluid is higher than the pH in human plasma (Jebens et al., “On theviscosity and pH of synovial fluid and the pH of blood,” The Journal ofBone and Joint Surgery, vol. 41 B, pages 388-400, 1959; Farr et al.,“Significance of the hydrogen ion concentration in synovial fluid inRheumatoid Arthritis,” Clinical and Experimental Rheumatology, vol. 3,pages 99-104, 1985).

Thus, the synovial fluid has several physiological conditions that aredifferent from those of the other parts of body, such as thephysiological conditions in the blood plasma. The synovial fluid has apH that is higher than other parts of the body, especially the bloodplasma. The synovial fluid has a lower concentration of glucose thanother parts of the body, such as blood plasma. The synovial fluid alsohas a lower concentration of protein than other parts of the body, suchas blood plasma.

Several antibodies have been used to treat joint disease by introducingthe antibodies into the synovial fluid. For example, the synovial fluidin an injured joint is known to contain many factors which have aninfluence on the progression of osteoarthritis (see, for example,Fernandes, et al., “The Role of Cytokines in OsteoarthritisPathophysiology”, Biorheology, vol. 39, pages 237-246, 2002). Cytokines,such as Interleukin-1 (IL-I) and Tumor Necrosis Factor-α (TNF-α), whichare produced by activated synoviocytes, are known to upregulate matrixmetalloproteinase (MMP) gene expression. Upregulation of MMP leads todegradation of the matrix and non-matrix proteins in the joints.Antibodies that neutralize cytokines may stop the progression ofosteoarthritis.

Using antibodies as drug is a promising strategy for the treatment ofjoint diseases. For example, antibodies (such as antibody againstaggrecan or aggrecanase) have been developed to treat osteoarthritis,which has by far the greatest prevalence among joint diseases(WO1993/022429A1). An antibody against acetylated high-mobility groupbox 1 (HMGB1) has been developed for diagnosis or treatment of jointdiseases that are inflammatory, autoimmune, neurodegenerative ormalignant diseases/disorders, such as arthritis. This antibody may beused to detect the acetylated form of HMGB1 in synovial fluid (WO2011/157905A1). Another antibody (CD20 antibody) has also been developedto treat damage to connective tissue and cartilage of the joints.

However, the antigens of these antibodies are often expressed in otherparts of the body carrying important physiological functions. Antibodiesagainst these antigens, though efficacious in treating joint diseases,may also significantly interfere with the normal physiological functionsof these antigens in other parts of the body. Therefore, severe sideeffects may be experienced by patients. It is thus desirable to developtherapeutics, such as antibodies against cytokines or other antigensthat can preferentially bind to their antigens (proteins or othermacromolecules) at higher affinity in the synovial fluid, while notbinding or only weakly binding to the same antigens in other parts ofthe body in order to reduce side effects.

Such conditionally active biological proteins may be conditionallyactive antibodies. In some embodiments, the present invention alsoprovides conditionally active biological proteins that are proteinsother than antibodies. For example, a conditionally active immuneregulator may be developed by the present invention for preferentiallyregulating the immune response in the synovial fluid, which may less orno effect on the immune response at other parts of the body.

The conditionally active biological proteins may be conditionally activesuppressors of cytokine signaling (SOCS). Many of these SOCS areinvolved in inhibiting the JAK-STAT signaling pathway. The conditionallyactive suppressors of cytokine signaling can preferentially suppress thecytokine signaling in the synovial fluid, while not or to a lesserextent suppressing the cytokine signaling in other parts of the body.

In some embodiments, the present invention provides a conditionallyactive biological protein derived from a wild-type biological protein.The conditionally active biological protein has a lower activity underat least one physiological condition in certain parts of the body suchas in blood plasma than the wild-type biological protein, and has ahigher activity than the wild-type biological protein under at least onephysiological condition in the synovial fluid. Such conditionally activebiological proteins can preferentially function in the synovial fluid,but not or to a lesser extent act upon other parts of the body.Consequently, such conditionally active biological proteins may havereduced side effects.

In some embodiments, the conditionally active biological proteins areantibodies against an antigen in or exposed to synovial fluid. Suchantigens may be any proteins involved in immune response/inflammation ina joint disease, though the antigen is often a cytokine. Theconditionally active antibody has a lower affinity to the antigen thanthe wild-type antibody for the same antigen under at least onephysiological condition in other parts of the body (such as bloodplasma), while has higher affinity for the antigen than the wild-typeantibody under at least one physiological condition of synovial fluid.Such conditionally active antibodies can bind weakly or not at all tothe antigen in other parts of the body, but bind, for example bindstrongly and tightly or bind stronger to the antigen in synovial fluid.

Conditionally Active Biological Proteins for Tumors

Cancer cells in a solid tumor are able to form a tumor microenvironmentin their surroundings to support the growth and metastasis of the cancercells. A tumor microenvironment is the cellular environment in which thetumor exists, including surrounding blood vessels, immune cells,fibroblasts, other cells, soluble factors, signaling molecules, anextracellular matrix, and mechanical cues that can promote neoplastictransformation, support tumor growth and invasion, protect the tumorfrom host immunity, foster therapeutic resistance, and provide nichesfor dormant metastases to thrive. The tumor and its surroundingmicroenvironment are closely related and interact constantly. Tumors caninfluence their microenvironment by releasing extracellular signals,promoting tumor angiogenesis and inducing peripheral immune tolerance,while the immune cells in the microenvironment can affect the growth andevolution of cancerous cells. See Swarts et al. “Tumor MicroenvironmentComplexity: Emerging Roles in Cancer Therapy,” Cancer Res, vol., 72,pages 2473-2480, 2012.

The tumor microenvironment is often hypoxic. As the tumor massincreases, the interior of the tumor grows farther away from existingblood supply, which leads to difficulties in fully supplying oxygen tothe tumor microenvironment. The partial oxygen pressure in the tumorenvironment is below 5 mm Hg in more than 50% of locally advanced solidtumors, in comparison with a partial oxygen pressure at about 40 mm Hgin blood plasma. In contrast, other parts of the body are not hypoxic.The hypoxic environment leads to genetic instability, which isassociated with cancer progression, via downregulating nucleotideexcision repair and mismatch repair pathways. Hypoxia also causes theupregulation of hypoxia-inducible factor 1 alpha (HIF1-α), which inducesangiogenesis, and is associated with poorer prognosis and the activationof genes associated with metastasis. See Weber et al., “The tumormicroenvironment,” Surgical Oncology, vol. 21, pages 172-177, 2012 andBlagosklonny, “Antiangiogenic therapy and tumor progression,” CancerCell, vol. 5, pages 13-17, 2004.

In addition, tumor cells tend to rely on energy generated from lacticacid fermentation, which does not require oxygen. So tumor cells areless likely to use normal aerobic respiration that does require oxygen.A consequence of using lactic acid fermentation is that the tumormicroenvironment is acidic (pH 6.5-6.9), in contrast to other parts ofthe body which are typically either neutral or slightly basic. Forexample, human blood plasma has a pH of about 7.4. See Estrella et al.,“Acidity Generated by the Tumor Microenvironment Drives Local Invasion,”Cancer Research, vol. 73, pages 1524-1535, 2013. The nutrientavailability in the tumor microenvironment is also low due to therelatively high nutrient demand of the proliferating cancer cells, incomparison with cells located in other parts of the body.

Further, the tumor microenvironment also contains many distinct celltypes not commonly found in other parts of the body. These cell typesinclude endothelial cells and their precursors, pericytes, smooth musclecells, Wbroblasts, carcinoma-associated Wbroblasts, myoWbroblasts,neutrophils, eosinophils, basophils, mast cells, T and B lymphocytes,natural killer cells and antigen presenting cells (APC) such asmacrophages and dendritic cells (Lorusso et al., “The tumormicroenvironment and its contribution to tumor evolution towardmetastasis,” Histochem Cell Biol, vol. 130, pages 1091-1103, 2008).

Accordingly, the tumor microenvironment has at least severalphysiological conditions that are different from those of other parts ofbody, such as the physiological conditions in blood plasma. The tumormicroenvironment has a pH (acidic) that is lower than other parts of thebody, especially the blood plasma (pH 7.4). The tumor microenvironmenthas a lower concentration of oxygen than other parts of the body, suchas blood plasma. Also, the tumor microenvironment has a lower nutrientavailability than other parts of the body, especially the blood plasma.The tumor microenvironment also has some distinct cell types that arenot commonly found in other parts of the body, especially the bloodplasma.

Some cancer drugs include antibodies that can penetrate into the tumormicroenvironment and act upon the cancer cells therein. Antibody-basedtherapy for cancer is well established and has become one of the mostsuccessful and important strategies for treating patients withhaematological malignancies and solid tumors. There is a broad array ofcell surface antigens that are expressed by human cancer cells that areoverexpressed, mutated or selectively expressed in cancer cells comparedwith normal tissues. These cell surface antigens are excellent targetsfor antibody cancer therapy.

Cancer cell surface antigens that may be targeted by antibodies fallinto several different categories. Haematopoietic differentiationantigens are glycoproteins that are usually associated with clusters ofdifferentiation (CD) groupings and include CD20, CD30, CD33 and CD52.Cell surface differentiation antigens are a diverse group ofglycoproteins and carbohydrates that are found on the surface of bothnormal and tumor cells. Antigens that are involved in growth anddifferentiation signaling are often growth factors and growth factorreceptors. Growth factors that are targets for antibodies in cancerpatients include CEA2, epidermal growth factor receptor (EGFR; alsoknown as ERBB1)12, ERBB2 (also known as HER2)13, ERBB3 (REF. 18), MET(also known as HGFR)19, insulin-like growth factor 1 receptor (IGF1R)20,ephrin receptor A3 (EPHA3)21, tumor necrosis factor (TNF)-relatedapoptosis-inducing ligand receptor 1 (TRAILR1; also known as TNFRSF10A),TRAILR2 (also known as TNFRSF10B) and receptor activator of nuclearfactor-κB ligand (RANKL; also known as TNFSF11)22. Antigens involved inangiogenesis are usually proteins or growth factors that support theformation of new microvasculature, including vascular endothelial growthfactor (VEGF), VEGF receptor (VEGFR), integrin αVβ3 and integrin α5β1(REF. 10). Tumor stroma and the extracellular matrix are indispensablesupport structures for a tumor. Stromal and extracellular matrixantigens that are therapeutic targets include fibroblast activationprotein (FAP) and tenascin. See Scott et al., “Antibody therapy ofcancer,” Nature Reviews Cancer, vol. 12, pages 278-287, 2012.

In addition to antibodies, other biological proteins have also shownpromise in treating cancers. Examples include tumor suppressors such asRetinoblastoma protein (pRb), p53, pVHL, APC, CD95, ST5, YPEL3, ST7, andST14. Some proteins that induce apoptosis in cancer cells may also beintroduced into tumors for shrinking the size of tumors. There are atleast two mechanisms that can induce apoptosis in tumors: the tumornecrosis factor-induced mechanism and the Fas-Fas ligand-mediatedmechanism. At least some of the proteins involved in either of the twoapoptotic mechanisms may be introduced to tumors for treatment.

Cancer stem cells are cancer cells that have the ability to give rise toall cell types found in a particular cancer sample, and are thereforetumor-forming. They may generate tumors through the stem cell processesof self-renewal and differentiation into multiple cell types. It isbelieved that cancer stem cells persist in tumors as a distinctpopulation and cause relapse and metastasis by giving rise to newtumors. Development of specific therapies targeted at cancer stem cellsmay improve the survival and quality of life of cancer patients,especially for sufferers of metastatic disease.

These drugs for treating tumors often interfere with normalphysiological functions in other parts of the body besides tumors. Forexample, proteins inducing apoptosis in tumors may also induce apoptosisin some other parts of the body thus causing side effects. Inembodiments where an antibody is used to treat tumors, the antigen ofthe antibody may also be expressed in other parts of the body where theyperform normal physiological functions. For example, monoclonal antibodybevacizumab (targeting vascular endothelial growth factor) to stop tumorblood vessel growth. This antibody can also prevent blood vessel growthor repair in other parts of the body, thus causing bleeding, poor woundhealing, blood clots, and kidney damage. Development of a conditionallyactive biological protein that concentrates on targeting mainly orsolely tumors is highly desirable for more effective tumor therapies.

In some embodiments, the present invention provides a conditionallyactive biological protein generated from a wild-type biological proteinthat may be a candidate for tumor treatment. The conditionally activebiological protein has lower activity under at least one physiologicalcondition in parts of the body other than the tumor microenvironmentsuch as blood plasma than the wild-type biological protein, while it hashigher activity under at least one physiological condition in the tumormicroenvironment than the wild-type biological protein. Suchconditionally active biological proteins can preferentially act uponcancer cells in the tumor microenvironment for treating tumors, and thuswill be less likely to cause side effects. In the embodiment where thebiological protein is an antibody against an antigen on the surface ofthe tumor cells where the antigen is exposed to the tumormicroenvironment, the conditionally active antibody has lower affinityto the antigen than the wild-type antibody in other parts of the body,e.g. a non-tumor microenvironment, while it has higher affinity to theantigen than the wild-type antibody in the tumor microenvironment. Suchconditionally active antibodies can bind weakly or not at all to theantigen in other parts of the body, but have greater binding, or bindstrongly and tightly, to the antigen in the tumor microenvironment.

In some embodiments, the conditionally active antibody is an antibodyagainst an immune checkpoint protein, resulting in inhibition of theimmune checkpoints. Such conditionally active antibodies have anincreased binding affinity to the immune checkpoint protein in a tumormicroenvironment in comparison to the wild-type antibody from which theconditionally active antibody is derived, and a decreased bindingaffinity to the immune checkpoint protein in a non-tumormicroenvironment in comparison to the wild-type antibody from which theconditionally active antibody is derived.

The immune checkpoints function as endogenous inhibitory pathways forthe immune system to maintain self-tolerance and modulate the durationand extent of immune response to antigenic stimulation, i.e., foreignmolecules, cells and tissues See Pardoll, Nature Reviews Cancer, vol.12, pages 252-264, 2012. Inhibition of immune checkpoints by suppressingone or more checkpoint proteins can cause super-activation of the immunesystem, especially T-cells, thus inducing the immune system to attacktumors. Checkpoint proteins suitable for the present invention includeCTLA4 and its ligands CD80 and CD86, PD1 and its ligands PDL1 and PDL2,T cell immunoglobulin and mucin protein-3 (TIM3) and its ligand GALS, Band T lymphocyte attenuator (BTLA) and its ligand HVEM (herpesvirusentry mediator), receptors such as killer cell immunoglobulin-likereceptor (KIR), lymphocyte activation gene-3 (LAG3) and adenosine A2areceptor (A2aR), as well as ligands B7-H3 and B7-H4. Additional suitableimmune checkpoint proteins are described in Pardoll, Nature ReviewsCancer, vol. 12, pages 252-264, 2012 and Nirschl & Drake, Clin CancerRes, vol. 19, pages 4917-4924, 2013, the disclosures of which are herebyincorporated herein by reference.

CTLA-4 and PD1 are two of the best known immune checkpoint proteins.CTLA-4 can down-regulate pathways of T-cell activation (Fong et al.,Cancer Res. 69(2):609-615, 2009; and Weber, Cancer Immunol. Immunother,58:823-830, 2009). Blockading CTLA-4 has been shown to augment T-cellactivation and proliferation. Inhibitors of CTLA-4 include anti-CTLA-4antibodies. Anti-CTLA-4 antibodies bind to CTLA-4 and block theinteraction of CTLA-4 with its ligands CD80 or CD86 thereby blocking thedown-regulation of the immune responses elicited by the interaction ofCTLA-4 with its ligand.

The checkpoint protein PD1 is known to suppress the activity of T cellsin peripheral tissues at the time of an inflammatory response toinfection and to limit autoimmunity. An in vitro PD1 blockade canenhance T-cell proliferation and cytokine production in response tostimulation by specific antigen targets or by allogeneic cells in mixedlymphocyte reactions. A strong correlation between PD1 expression andreduced immune response was shown to be caused by the inhibitoryfunction of PD1, i.e., by inducing immune checkpoints (Pardoll, NatureReviews Cancer, 12: 252-264, 2012). A PD1 blockade can be accomplishedby a variety of mechanisms including antibodies that bind PD1 or itsligands, PDL1 or PDL2.

Past research has discovered antibodies against several checkpointproteins (CTLA4, PD1, PD-L1). These antibodies are effective in treatingtumors by inhibiting the immune checkpoints thereby super-activating theimmune system, especially the T-cells, for attacking tumors (Pardoll,Nature Reviews Cancer, vol. 12, pages 252-264, 2012). However, thesuper-activated T-cells may also attack host cells and/or tissues,resulting in collateral damage to a patient's body. Thus, therapy basedon use of these known antibodies for inhibition of immune checkpoints isdifficult to manage and the risk to the patient is a serious concern.For example, an FDA approved antibody against CTLA-4 carries a black boxwarning due to its high toxicity.

The present invention addresses the problem of collateral damage bysuper-activated T-cells by providing conditionally active antibodiesagainst immune checkpoint proteins. These conditionally activeantibodies preferentially activate the immune checkpoints in atumor-microenvironment. At the same time, the immune checkpoints in thenon-tumor-microenvironment(s), e.g. normal body tissue, are notinhibited or are less inhibited by the conditionally active antibodiessuch that in the non-tumor microenvironment the potential for collateraldamage to the body is reduced. This goal is achieved by engineering theconditionally active antibody to be more active in the tumormicroenvironment than in the non-tumor microenvironment.

In some embodiments, the conditionally active antibody against an immunecheckpoint protein may have a ratio of binding activity to an immunecheckpoint protein in the tumor-microenvironment to the binding activityto the same immune checkpoint protein in a non-tumor microenvironment ofat least about 1.1, or at least about 1.2, or at least about 1.4, or atleast about 1.6, or at least about 1.8, or at least about 2, or at leastabout 2.5, or at least about 3, or at least about 5, or at least about7, or at least about 8, or at least about 9, or at least about 10, or atleast about 15, or at least about 20. A typical assay for measuring thebinding activity of an antibody is an ELISA assay.

Highly immunogenic tumors, such as malignant melanoma, are mostvulnerable to a super-activated immune system achieved by immune systemmanipulation. Thus the conditionally active antibodies against immunecheckpoint proteins may be especially effective for treating such highlyimmunogenic tumors. However, other types of tumors are also vulnerableto a super-activated immune system.

In some embodiments, the conditionally active antibodies against theimmune checkpoint proteins may be used in combination therapy. Forexample, combination therapy may include a conditionally active antibodyagainst a tumor cell surface molecule (tumor specific antigen) and aconditionally active antibody against an immune checkpoint protein. Inone embodiment, both the binding activity of the conditionally activeantibody to the tumor cell surface molecule and the binding activity ofthe conditionally active antibody to the immune checkpoint protein mayreside in a single protein, i.e., a bispecific conditionally activeantibody as disclosed herein. In some further embodiments, combinationtherapy may include a conditionally active antibody against a tumor cellsurface molecule (tumor specific antigen) and two or more conditionallyactive antibodies against two or more different immune checkpointproteins. In one embodiment, all of these binding activities may residein a single protein, i.e., a multispecific antibody as disclosed herein.

Since the conditionally active antibodies are more active in a tumormicroenvironment in comparison with the activity of the wild-typeantibody against the same tumor cell surface molecule or checkpointprotein from which the conditionally active antibody is derived, thesecombination therapies can provide both an enhanced efficacy and asignificant reduction in toxicity. The reduced toxicity of theseconditionally active antibodies, especially the antibodies against theimmune checkpoint proteins, can allow safe use of potent antibodies,such as ADC antibodies as described herein, as well as a higher dose ofthe antibodies.

In some embodiments, the conditionally active antibodies against thecheckpoint proteins may be in a prodrug form. For example, theconditionally active antibodies may be prodrugs that have no desireddrug activity before being cleaved and turned into a drug form. Theprodrugs may be cleaved preferentially in a tumor-microenvironment,either because the enzyme that catalyzes such cleavage existspreferentially in the tumor-microenvironment or because theconditionally active antibodies make the cleavage site more accessiblein a tumor microenvironment, in comparison with the accessibility of thecleavage site in a non-tumor microenvironment. Conditionally activebiological proteins for stem cell niches, including tumor stem cells

Stem cells exist in an environment called stem cell niche in the body,which constitutes a basic unit of tissue physiology, integrating signalsthat mediate the response of stem cells to the needs of organisms. Yetthe niche may also induce pathologies by imposing aberrant functions onstem cells or other targets. The interplay between stem cells and theirniches creates the dynamic system necessary for sustaining tissues, andfor the ultimate design of stem-cell therapeutics (Scadden, “Thestem-cell niche as an entity of action,” Nature, vol. 441, pages1075-1079, 2006). Common stem cell niches in vertebrates include thegermline stem cell niche, the hematopoietic stem cell niche, the hairfollicle stem cell niche, the intestinal stem cell niche, and thecardiovascular stem cell niche.

The stem cell niche is a specialized environment that is different fromother parts of the body (e.g. blood plasma) (Drummond-Barbosa, “StemCells, Their Niches and the Systemic Environment: An Aging Network,”Genetics, vol. 180, pages 1787-1797, 2008; Fuchs, “Socializing with theNeighbors: Stem Cells and Their Niche,” Cell, vol. 116, pages 769-778,2004). The stem cell niche is hypoxic where oxidative DNA damage isreduced. Direct measurements of oxygen levels have revealed that bonemarrow is, in general, quite hypoxic (˜1%-2% O2), in comparison to bloodplasma (Keith et al., “Hypoxia-Inducible Factors, Stem Cells, andCancer,” Cell, vol. 129, pages 465-472, 2007; Mohyeldin et al., “Oxygenin Stem Cell Biology: A Critical Component of the Stem Cell Niche,” CellStem Cell, vol. 7, pages 150-161, 2010). In addition, the stem cellniches need to have several other factors to regulate stem cellcharacteristics within the niches: extracellular matrix components,growth factors, cytokines, and factors of the physiochemical nature ofthe environment including the pH, ionic strength (e.g. Ca²⁺concentration) and metabolites.

Accordingly, the stem cell niche has at least several physiologicalconditions that are different from those of the other parts of body,such as the physiological conditions in the blood plasma. The stem cellniche has a lower oxygen concentration (1-2%) than other parts of thebody, especially the blood plasma. Other physiological conditions forthe stem cell niche including pH and ionic strength, may also bedifferent from other parts of the body.

Stem cell therapy is an interventional strategy that introduces newadult stem cells into damaged tissue in order to treat disease orinjury. This strategy depends on the ability of stem cells to self-renewand give rise to subsequent offspring with variable degrees ofdifferentiation capacities. Stem cell therapy offers significantpotential for regeneration of tissues that can potentially replacediseased and damaged areas in the body, with minimal risk of rejectionand side effects. Therefore, delivering a drug (biological protein (e.g.antibody) or chemical compound) to the stem cell niche for influencingthe renewal and differentiation of stem cells is an important part ofstem cell therapy.

There are several examples on how the stem cell niches influence therenewal and/or differentiation of the stem cells in mammals. The firstis in the skin, where the β-1 integrin is known to be differentiallyexpressed on primitive cells and to participate in constrainedlocalization of a stem-cell population through interaction with matrixglycoprotein ligands. Second, in the nervous system, the absence oftenascin C alters neural stem-cell number and function in thesubventricular zone. Tenascin C seems to modulate stem-cell sensitivityto fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 4(BMP4), resulting in increased stem-cell propensity. Third, anothermatrix protein, the Arg-Gly-Asp-containing sialoprotein, osteopontin(OPN), has now been demonstrated to contribute to haematopoietic stemcell regulation. OPN interacts with several receptors known to be onhaematopoietic stem cells, CD44, and α4 and α5β1 integrins. OPNproduction can vary markedly, particularly with osteoblast activation.Animals deficient in OPN have an increased HS-cell number, because alack of OPN leads to superphysiologic stem-cell expansion understimulatory conditions. Therefore, OPN seems to serve as a constraint onhaematopoietic stem cell numbers, limiting the number of stem cellsunder homeostatic conditions or with stimulation. See Scadden, “Thestem-cell niche as an entity of action,” Nature, vol. 441, pages1075-1079, 2006.

Xie et al. “Autocrine signaling based selection of combinatorialantibodies that transdifferentiate human stem cells,” Proc Natl Acad SciUSA, vol. 110, pages 8099-8104, 2013) discloses a method of usingantibodies to influence stem cell differentiation. The antibodies areagonists for a granulocyte colony stimulating factor receptor. Unlikethe natural granulocyte-colony stimulating factor that activates cellsto differentiate along a predetermined pathway, the isolated agonistantibodies transdifferentiated human myeloid lineage CD34+ bone marrowcells into neural progenitors. Melidoni et al. (“Selecting antagonisticantibodies that control differentiation through inducible expression inembryonic stem cells,” Proc Natl Acad Sci USA, vol. 110, pages17802-17807, 2013) also discloses a method of using an antibody tointerfere the interaction between FGF4 and its receptor FGFR1β,therefore block the autocrine FGF4-mediated embryonic stem celldifferentiation.

Knowledge of the functions of ligands/receptors in stem celldifferentiation has enabled the strategy of applying biological proteinsto interfere with these ligands/receptors for the purpose of regulatingor even directing stem cell differentiation. The ability to controldifferentiation of genetically unmodified human stem cells through theadministration of antibodies into the stem cell niche can provide new exvivo or in vivo approaches to stem cell-based therapeutics. In someembodiments, the present invention provides a conditionally activebiological protein generated from a wild-type biological protein that iscapable of entering the stem cell niches, including cancer stem cells,to regulate stem cell or tumor development. The conditionally activebiological protein has lower activity than the wild-type biologicalprotein under at least one physiological condition in other parts of thebody, while it has higher activity than the wild-type biological proteinunder at least one physiological condition in the stem cell niche, forexample the cancer stem cell environment. Such conditionally activebiological proteins will be less likely to cause side effects andpreferentially act in the stem cell niche to regulate renewal anddifferentiation of stem cells. In some embodiments, the conditionallyactive biological proteins are antibodies. Such conditionally activeantibodies can bind weakly or not at all to their antigens in otherparts of the body, but bind strongly and tightly to the antigens in thestem cell niche.

The conditionally active proteins for the synovial fluid, tumormicroenvironment and stem cell niches of the present invention aregenerated by a method for evolving a DNA that encodes a wild-typebiological protein to create a mutant DNA library. The mutant DNAlibrary is then expressed to obtain mutant proteins. The mutant proteinsare screened for a conditionally active biological protein that has ahigher activity than the wild-type biological protein under at least onephysiological condition of a first part of the body selected from thegroup consisting of synovial fluid, tumor microenvironment, and stemcell niches, and has lower activity than the wild-type biologicalprotein under at least one physiological condition at a second part ofthe body that is different from the first part of the body. The secondpart of the body may be the blood plasma. Such selected mutantbiological proteins are conditionally active biological proteins thathave high activity in the first part of the body but low activity in thesecond parts of the body.

Such conditionally active biological proteins are advantageous inlowering side effects of the wild-type protein, since the conditionallyactive biological protein has lower activity in the other parts of thebody where the conditionally active biological protein is not intendedto act. For instance, if the conditionally active biological protein isintended to be introduced into the tumor microenvironment, the fact thatthe conditionally active biological protein has low activity in parts ofthe body other than the tumor microenvironment means such conditionallyactive biological protein will be less likely to interfere with normalphysiological functions in parts of the body other than the tumormicroenvironment. At the same time, the conditionally active biologicalprotein has high activity in the tumor microenvironment, which gives theconditionally active biological protein a higher efficacy in treatingtumors.

Because of the reduced side effects, the conditionally active biologicalprotein will allow a significantly higher dose of the protein to besafely used, in comparison with the wild-type biological protein. Thisis especially beneficial for an antibody against a cytokine or a growthfactor, because antibodies against the cytokine or growth factor mayinterfere with normal physiological functions of the cytokine or growthfactor in other parts of the body. By using a conditionally activebiological protein, with reduced side effects, higher doses may be usedto achieve higher efficacy.

The conditionally active biological proteins for acting in one of asynovial fluid, tumor microenvironment, or stem cell niche can alsoenable new drug targets to be used. Using traditional biologicalproteins as therapeutics may cause unacceptable side effects. Forexample, inhibition of an epidermal growth factor receptor (EGFR) canvery effectively suppress tumor growth. However, a drug inhibiting EGFRwill also suppress growth at the skin and gastrointestinal (GI) tract.The side effects render EGFR unsuitable as a tumor drug target. Using aconditionally active antibody that binds to EGFR at high affinity inonly the tumor microenvironment, but not or at very low affinity at anyother parts of the body, will significantly reduce the side effects andat the same time suppress tumor growth. In this case, EGFR may become aneffective new tumor drug target by using conditionally activeantibodies.

In another example, suppressing cytokines is often beneficial inrepairing joint damage. However, suppressing cytokines in other parts ofthe body also may suppress the immune response of the body, causing animmune deficiency. Thus, cytokines in synovial fluid are not idealtargets for developing traditional antibody drugs for treatment of jointdamage. However, by using conditionally active antibodies thatpreferentially bind to cytokines in the synovial fluid, while not oronly weakly to the same cytokines in other parts of the body, the sideeffect of immune deficiency can be dramatically reduced. Therefore,cytokines in synovial fluid may become suitable targets for repairingjoint damage by using conditionally active antibodies.

Conditionally Active Viral Particles

Viral particles have long been used as delivery vehicles fortransporting proteins, nucleic acid molecules, chemical compounds orradioactive isotopes to a target cell or tissue. Viral panicles that arecommonly used as delivery vehicles include retoviruses, adenoviruses,lentivirus, herpes virus, and adeno-associated viruses. The viralparticles recognize their target cells through a surface protein thatserves as a recognition protein for specific binding to a cellularprotein that serves as target protein of the target cells, often in aligand-receptor binding system (Lentz, “The recognition event betweenvirus and host cell receptor: a target for antiviral agents,” J. of Gen.Virol., vol. 71, pages 751-765, 1990, incorporated herein by reference).For example, the viral recognition protein may be a ligand for areceptor on the target cells. The specificity between a ligand and areceptor allows the viral particles to specifically recognize anddeliver their content to a target cell.

Techniques for developing artificial viral particles from wild-typeviruses are well known to a person skilled in the art. Known artificialviral particles as delivery vehicles include these based on retroviruses(see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S.Pat. No. 5,219,740; WO 93/11230; WO 93/10218; U.S. Pat. No. 4,777,127;GB Patent No, 2,200,651; EP 0 345 242; and WO 91/02805), alphavirusSindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247),Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equineencephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCCVR-532)), and adeno-associated viruses (see, e.g., WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655).

Generally, the artificial viral particles are constructed by inserting aforeign recognition protein into a virus particle, often replacing thenative recognition protein by recombinant technology. The foreignrecognition protein may be, for example, an antibody, a receptor, aligand or a collagen binding domain. The present invention provides aconditionally active recognition protein that is inactive or less activefor binding to a cell at a normal physiological condition, and that isactive or more active for binding to a cell at an aberrant condition.The conditionally active recognition protein can thereby preferentiallybind to target cells of diseased tissue and/or at a disease site basedon the presence of an abnormal condition at that site and avoid or onlyminimally bind to the cells of normal tissue where a normalphysiological condition exists. The conditionally active recognitionprotein may be expressed and displayed on the surface of a viralparticle.

In some embodiments, the present invention provides a method of evolvinga wild-type recognition protein and screening for a conditionally activerecognition protein. The conditionally active recognition protein isless active in binding to a cell than the wild-type recognition proteinunder a normal physiological condition, and more active in binding to acell than the wild-type recognition protein under an aberrant condition.Such a conditionally active recognition protein may be inserted into aviral particle by well-known recombinant technology to generate aconditionally active viral particle.

In another embodiment, the present invention provides a conditionallyactive viral particle comprising a conditionally active recognitionprotein, which allows the conditionally active viral particle torecognize and bind with the target cells of diseased tissue or at adisease site, but not the cells of normal tissue. Such a conditionallyactive viral particle can preferentially deliver therapeutics within theviral particle to the disease tissue or disease site, while theconditionally active viral particle delivers less or does not deliverthe therapeutics to the cells of normal tissue.

In some embodiments, the target cells at a disease site are inside azone or microenvironment with an abnormal pH (e.g., pH 6.5) or anabnormal temperature, in comparison with the pH or temperature in otherparts of the body that are healthy or not suffering from the particulardisease or disease state. In this embodiment, the conditionally activerecognition protein is less active than a wild-type recognition proteinin binding with a target protein of a target cell at a normalphysiological pH or temperature, and more active than a wild-typerecognition protein in binding with the target protein of a target cellat an abnormal pH or temperature. In this manner, the recognitionprotein will preferentially bind at a site where an abnormal pH ortemperature is encountered thereby delivering a treatment to the site ofa disease.

In one embodiment, the viral particle may comprise a conditionallyactive antibody of the present invention, and especially the variableregion of an antibody (e.g., Fab, Fab′, Fv). Such a conditionally activeantibody can bind to the target protein (as antigen) of a target cellwith lower affinity than a wild-type antibody under a normalphysiological condition which may be encountered at a location withnormal tissue, and a higher affinity than the wild-type antibody underaberrant condition which may be encountered at a disease site ordiseased tissue. The conditionally active antibody may be derived fromthe wild-type antibody according to the method of the present invention.

In an embodiment, the target protein on the target cell includestyrosine kinase growth factor receptors which are overexpressed on thecell surfaces in, for example, many tumors. Exemplary tyrosine kinasegrowth factors are VEGF receptors, FGF receptors, PDGF receptors, IGFreceptors, EGF receptors, TGF-alpha receptors, TGF-beta receptors,HB-EGF receptors, ErbB2 receptors, ErbB3 receptors, and ErbB4 receptors.Conditionally active DNA/RNA modifying proteins

DNA/RNA modifying proteins have been discovered as a form of newgenome-engineering tools, particularly one called CRISPR, which canallow researchers to perform microsurgery on genes, precisely and easilychanging a DNA sequence at exact locations on a chromosome (genomeediting, Mali et al., “Cas9 as a versatile tool for engineeringbiology,” Nature Methods, vol. 10, pages 957-963, 2013). For example,sickle-cell anemia is caused by a single base mutation, which canpotentially be corrected using DNA/RNA modifying proteins. Thetechnology may precisely delete or edit bits of a chromosome, even bychanging a single base pair (Makarova et al., “Evolution andclassification of the CRISPR-Cas systems,” Nature Reviews Microbiology,vol. 9, pages 467-477, 2011).

Genome editing with CRISPR has the ability to quickly and simultaneouslymake multiple genetic changes to a cell. Many human illnesses, includingheart disease, diabetes, and neurological diseases, are affected bymutations in multiple genes. This CRISPR-based technology has thepotential to reverse the disease causing mutations and cure thesediseases or at least reduce the severity of these diseases. Genomeediting relies on CRISPR associated (Cas) proteins (a family of enzymes)for cutting the genomic DNA. Typically, the Cas protein is guided by asmall guide RNA to a targeted region in the genome, where the guide RNAmatches the target region. Because the Cas protein has little or nosequence specificity, the guide RNA serves as a pointer for the Casprotein to achieve precise genome editing. In one embodiment, one Casprotein may be used with multiple guide RNAs to simultaneously correctmultiple gene mutations.

There are many Cas proteins. Examples include Cas1, Cas2, Cas3′, Cas3″,Cas4, Cas5, Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c,Cas9, Cas10, Cas10d, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5,Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1,Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1,Csf2, Csf3, and Csf4 ((Makarova et al., “Evolution and classification ofthe CRISPR-Cas systems,” Nature Reviews Microbiology, vol. 9, pages467-477, 2011).

To conduct genome editing, the Cas protein has to enter the target cell.Cells in a subject may have a different intracellular pH inside of thecells. Some cells in diseased tissue have an abnormal intracellular pH.For example, some tumor cells tend to have an alkaline intracellular pHof about 7.12-7.65, while cells in normal tissue have a neutralintracellular pH ranging from 6.99-7.20. See Cardone et al., “The roleof disturbed pH dynamics and the Na(+)/H(+) exchanger in metatasis,”Nat. Rev. Cancer, vol. 5, pages 786-795, 2005. In chronic hypoxia, thecells in diseased tissue have an intracellular pH of about 7.2-7.5, alsohigher than the intracellular pH of normal tissue (Rios et al., “Chronichypoxia elevates intracellular pH and activates Na+/H+ exchange inpulmonary arterial smooth muscle cells,” American Journal ofPhysiology—Lung Cellular and Molecular Physiology, vol. 289, pagesL867-L874, 2005). Further, in ischemia cells, the intracellular pH istypical! in a range of 6.55-6.65, which is lower than the intracellularpH of normal tissue (Haqberg, “Intracellular pH during ischemic inskeletal muscle: relationship to membrane potential, extracellular pH,tissue lactic acid and ATP,” Pflugers Arch., vol. 404, pages 342-347,1985). More examples of abnormal intracellular pH in diseased tissue arediscussed in Han et al., “Fluorescent Indicators for Intracellular pH,”Chem Rev., vol. 110, pages 2709-2728, 2010.

The present invention provides a method for producing a conditionallyactive Cas protein from a wild-type Cas protein, where the conditionallyactive Cas protein has a decreased enzymatic activity relative to theactivity of the wild-type Cas protein under a normal physiologicalcondition inside a normal cell, and an increased enzymatic activityrelative to the activity of the wild-type Cas protein under an aberrantcondition inside a target cell such as one of the diseased cellsdiscussed above. In some embodiments, the normal physiological conditionis an intracellular pH about neutral, and the aberrant condition is adifferent intracellular pH that is above or below neutral. In anembodiment, the aberrant condition is an intracellular pH of from 7.2 to7.65 or an intracellular pH of from 6.5-6.8.

In some embodiments, the conditionally active Cas protein may bedelivered to a target cell using the conditionally active viral particleof the present invention. The conditionally active viral particlecomprises the conditionally active Cas protein and at least one guideRNA for directing the Cas protein to the location at which Cas proteinwill edit the genomic DNA.

Method of Generating Conditionally Active Biological Proteins

One or more mutagenesis techniques are employed to evolve the DNA whichencodes the wild-type protein to create a library of mutant DNA; themutant DNA is expressed to create a library of mutant proteins; and thelibrary is subjected to a screening assay under a normal physiologicalcondition and under one or more aberrant conditions. Conditionallyactive biologic proteins are selected from those proteins which exhibitboth (a) a decrease in activity in the assay at the normal physiologicalcondition compared to the wild-type protein, and (b) an increase inactivity in the assay under the aberrant condition compared to thewild-type protein. Alternatively, conditionally active biologic proteinsare selected from those proteins which exhibit changes in activity,reversibly or irreversibly, in two or more different physiologicalconditions. In some embodiments, the wild-type protein is an antibody.

Generation of Evolved Molecules from a Parent Molecule

Conditionally active proteins can be generated through a process ofmutagenesis and screening for individual mutations for a reduction inactivity at the wild-type condition with activity at non wild-typeconditions remaining the same or better than the activity at thewild-type condition.

The disclosure provides for a method for generating a nucleic acidvariant encoding a polypeptide having enzyme activity, wherein thevariant has an altered biological activity from that which naturallyoccurs, the method comprising (a) modifying the nucleic acid by (i)substituting one or more nucleotides for a different nucleotide, whereinthe nucleotide comprises a natural or non-natural nucleotide; (ii)deleting one or more nucleotides, (iii) adding one or more nucleotides,or (iv) any combination thereof. In one aspect, the non-naturalnucleotide comprises inosine. In another aspect, the method furthercomprises assaying the polypeptides encoded by the modified nucleicacids for altered enzyme activity, thereby identifying the modifiednucleic acid(s) encoding a polypeptide having altered enzyme activity.In one aspect, the modifications of step (a) are made by PCR,error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis, gene reassembly, gene sitesaturated mutagenesis, ligase chain reaction, in vitro mutagenesis,ligase chain reaction, oligonucleotide synthesis, any DNA-generatingtechnique and any combination thereof. In another aspect, the methodfurther comprises at least one repetition of the modification step (a).

The disclosure further provides a method for making a polynucleotidefrom two or more nucleic acids, the method comprising: (a) identifyingregions of identity and regions of diversity between two or more nucleicacids, wherein at least one of the nucleic acids comprises a nucleicacid of the disclosure; (b) providing a set of oligonucleotides whichcorrespond in sequence to at least two of the two or more nucleic acids;and, (c) extending the oligonucleotides with a polymerase, therebymaking the polynucleotide.

Any technique of mutagenesis can be employed in various embodiments ofthe disclosure. Stochastic or random mutagenesis is exemplified by asituation in which a parent molecule is mutated (modified or changed) toyield a set of progeny molecules having mutation(s) that are notpredetermined. Thus, in an in vitro stochastic mutagenesis reaction, forexample, there is not a particular predetermined product whoseproduction is intended; rather there is an uncertainty—hencerandomness—regarding the exact nature of the mutations achieved, andthus also regarding the products generated. Stochastic mutagenesis ismanifested in processes such as error-prone PCR and stochasticshuffling, where the mutation(s) achieved are random or notpredetermined. The variant forms can be generated by error-pronetranscription, such as an error-prone PCR or use of a polymerase whichlacks proof-reading activity (see, Liao (1990) Gene 88: 107-111), of thefirst variant form, or, by replication of the first form in a mutatorstrain (mutator host cells are discussed in further detail below, andare generally well known). A mutator strain can include any mutants inany organism impaired in the functions of mismatch repair. These includemutant gene products of mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC,umuD, sbcB, recJ, etc. The impairment is achieved by genetic mutation,allelic replacement, selective inhibition by an added reagent such as asmall compound or an expressed antisense RNA, or other techniques.Impairment can be of the genes noted, or of homologous genes in anyorganism.

Current mutagenesis methods in widespread use for creating alternativeproteins from a starting molecule are oligonucleotide-directedmutagenesis technologies, error-prone polymerase chain reactions(error-prone PCR) and cassette mutagenesis, in which the specific regionto be optimized is replaced with a synthetically mutagenizedoligonucleotide. In these cases, a number of mutant sites are generatedaround certain sites in the original sequence.

In oligonucleotide-directed mutagenesis, a short sequence is replacedwith a synthetically mutagenized oligonucleotide. Inoligonucleotide-directed mutagenesis, a short sequence of thepolynucleotide is removed from the polynucleotide using restrictionenzyme digestion and is replaced with a synthetic polynucleotide inwhich various bases have been altered from the original sequence. Thepolynucleotide sequence can also be altered by chemical mutagenesis.Chemical mutagens include, for example, sodium bisulfite, nitrous acid,hydroxylamine, hydrazine or formic acid. Other agents which areanalogues of nucleotide precursors include nitrosoguanidine,5-bromouracil, 2-aminopurine, or acridine. Generally, these agents areadded to the PCR reaction in place of the nucleotide precursor therebymutating the sequence. Intercalating agents such as proflavine,acriflavine, quinacrine and the like can also be used. Randommutagenesis of the polynucleotide sequence can also be achieved byirradiation with X-rays or ultraviolet light. Generally, plasmidpolynucleotides so mutagenized are introduced into E. coli andpropagated as a pool or library of hybrid plasmids.

Error-prone PCR uses low-fidelity polymerization conditions to introducea low level of point mutations randomly over a long sequence. In amixture of fragments of unknown sequence, error-prone PCR can be used tomutagenize the mixture.

In cassette mutagenesis, a sequence block of a single template istypically replaced by a (partially) randomized sequence. Reidhaar-OlsonJ F and Sauer R T: Combinatorial cassette mutagenesis as a probe of theinformational content of protein sequences. Science 241(4861):53-57,1988.

Alternatively, any technique of non-stochastic or non-random mutagenesiscan be employed in various embodiments of the disclosure. Non-stochasticmutagenesis is exemplified by a situation in which a parent molecule ismutated (modified or changed) to yield a progeny molecule having one ormore predetermined mutations. It is appreciated that the presence ofbackground products in some quantity is a reality in many reactionswhere molecular processing occurs, and the presence of these backgroundproducts does not detract from the non-stochastic nature of amutagenesis process having a predetermined product. Site-saturationmutagenesis and synthetic ligation reassembly are examples ofmutagenesis techniques where the exact chemical structure(s) of theintended product(s) are predetermined.

One method of site-saturation mutagenesis is disclosed in U.S. patentapplication publication 2009/0130718, which is incorporated herein byreference. This method provides a set of degenerate primerscorresponding to codons of a template polynucleotide, and performspolymerase elongation to produce progeny polynucleotides, which containsequences corresponding to the degenerate primers. The progenypolynucleotides can be expressed and screened for directed evolution.Specifically, this is a method for producing a set of progenypolynucleotides, comprising the steps of (a) providing copies of atemplate polynucleotide, each comprising a plurality of codons thatencode a template polypeptide sequence; and (b) for each codon of thetemplate polynucleotide, performing the steps of (1) providing a set ofdegenerate primers, where each primer comprises a degenerate codoncorresponding to the codon of the template polynucleotide and at leastone adjacent sequence that is homologous to a sequence adjacent to thecodon of the template polynucleotide; (2) providing conditions allowingthe primers to anneal to the copies of the template polynucleotides; and(3) performing a polymerase elongation reaction from the primers alongthe template; thereby producing progeny polynucleotides, each of whichcontains a sequence corresponding to the degenerate codon of theannealed primer; thereby producing a set of progeny polynucleotides.

Site-saturation mutagenesis relates to the directed evolution of nucleicacids and screening of clones containing the evolved nucleic acids forresultant activity(ies) of interest, such nucleic acid activity(ies)&/or specified protein, particularly enzyme, activity(ies) of interest.

Mutagenized molecules provided by this technique may have chimericmolecules and molecules with point mutations, including biologicalmolecules that contain a carbohydrate, a lipid, a nucleic acid, &/or aprotein component, and specific but non-limiting examples of theseinclude antibiotics, antibodies, enzymes, and steroidal andnon-steroidal hormones.

Site saturation mutagenesis relates generally to a method of: 1)preparing a progeny generation of molecule(s) (including a molecule thatis comprised of a polynucleotide sequence, a molecule that is comprisedof a polypeptide sequence, and a molecule that is comprised in part of apolynucleotide sequence and in part of a polypeptide sequence), that ismutagenized to achieve at least one point mutation, addition, deletion,&/or chimerization, from one or more ancestral or parental generationtemplate(s); 2) screening the progeny generation molecule(s)—preferablyusing a high throughput method—for at least one property of interest(such as an improvement in an enzyme activity or an increase instability or a novel chemotherapeutic effect); 3) optionally obtaining&/or cataloguing structural &/or and functional information regardingthe parental &/or progeny generation molecules; and 4) optionallyrepeating any of steps 1) to 3).

In site saturation mutagenesis, there is generated (e.g. from a parentpolynucleotide template)—in what is termed “codon site-saturationmutagenesis”—a progeny generation of polynucleotides, each having atleast one set of up to three contiguous point mutations (i.e. differentbases comprising a new codon), such that every codon (or every family ofdegenerate codons encoding the same amino acid) is represented at eachcodon position. Corresponding to—and encoded by—this progeny generationof polynucleotides, there is also generated a set of progenypolypeptides, each having at least one single amino acid point mutation.In a preferred aspect, there is generated—in what is termed “amino acidsite-saturation mutagenesis”—one such mutant polypeptide for each of the19 naturally encoded polypeptide-forming alpha-amino acid substitutionsat each and every amino acid position along the polypeptide. Thisyields—for each and every amino acid position along the parentalpolypeptide—a total of 20 distinct progeny polypeptides including theoriginal amino acid, or potentially more than 21 distinct progenypolypeptides if additional amino acids are used either instead of or inaddition to the 20 naturally encoded amino acids.

Other mutagenesis techniques can also be employed which involverecombination and more specifically a method for preparingpolynucleotides encoding a polypeptide by a method of in vivore-assortment of polynucleotide sequences containing regions of partialhomology, assembling the polynucleotides to form at least onepolynucleotide and screening the polynucleotides for the production ofpolypeptide(s) having a useful property.

In another aspect, mutagenesis techniques exploit the natural propertyof cells to recombine molecules and/or to mediate reductive processesthat reduce the complexity of sequences and extent of repeated orconsecutive sequences possessing regions of homology.

Various mutagenesis techniques can be used alone or in combination toprovide a method for generating hybrid polynucleotides encodingbiologically active hybrid polypeptides with enhanced activities. Inaccomplishing these and other objects, there has been provided, inaccordance with one aspect of the disclosure, a method for introducingpolynucleotides into a suitable host cell and growing the host cellunder conditions that produce a hybrid polynucleotide.

Chimeric genes have been made by joining 2 polynucleotide fragmentsusing compatible sticky ends generated by restriction enzyme(s), whereeach fragment is derived from a separate progenitor (or parental)molecule. Another example is the mutagenesis of a single codon position(i.e. to achieve a codon substitution, addition, or deletion) in aparental polynucleotide to generate a single progeny polynucleotideencoding for a single site-mutagenized polypeptide.

Further, in vivo site specific recombination systems have been utilizedto generate hybrids of genes, as well as random methods of in vivorecombination, and recombination between homologous but truncated geneson a plasmid. Mutagenesis has also been reported by overlappingextension and PCR.

Non-random methods have been used to achieve larger numbers of pointmutations and/or chimerizations, for example comprehensive or exhaustiveapproaches have been used to generate all the molecular species within aparticular grouping of mutations, for attributing functionality tospecific structural groups in a template molecule (e.g. a specificsingle amino acid position or a sequence comprised of two or more aminoacids positions), and for categorizing and comparing specific groupingof mutations.

Any of these or other methods of evolving can be employed in the presentdisclosure to generate a new population of molecules (library) from oneor more parent molecules.

Once formed, the constructs may, or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector, and transfected into an appropriate host cell.

In some embodiments, the evolving step is directed at the Fc region of awild-type antibody. In these embodiments, the Fc region of the wild-typeantibody is modified in the resultant conditionally active antibody. TheFc regions that may be modified include the Fc region of an antibody(e.g., in a full-length IgG antibody including full-length IgG1, IgG2,IgG3 or IgG4 antibodies, a chimeric antibody, or a humanized antibody),or in a fusion protein that contains a Fc region, or a part of a Fcregion (referred to as an “immunoglobulin (Ig) fusion protein”, “Fcfusion protein”, or “Fc fusion polypeptide”).

Modified Fc regions of antibodies have been described in the art,including in US2006/0104989. The modified Fc regions can have a singleamino acid substitution (also referred to as a Fc variant herein)relative to the sequence of a corresponding unmodified (wild-type orparent) Fc region, and may have one or more properties that differ froma corresponding wild-type or parent having an unmodified Fc region aswell as from other antibodies having modified Fc regions that have beendescribed in the art. Such properties may include, for example,increased binding to one or more Fc receptors and/or modified bindingunder different pH conditions.

The modified Fc regions can be incorporated into any antibody or Fcfusion polypeptide using standard molecular biology techniques, and allsuch modified antibodies and Fc fusion polypeptides are intended to beencompassed by the invention. Fc refers to the last two constant regionIg domains of IgA, IgD, and IgG, and the last three constant region Igdomains of IgE and IgM, and the flexible hinge N-terminal to thesedomains. For IgA and IgM, Fc may include the J chain. Fc is bound byreceptors, FcRs, which are present on certain cells. As the affinity ofthe interaction between Fc and certain FcRs present on particular cellscorrelates with targeted cytotoxicity, and clinical efficacy in humanscorrelates with the allotype of high or low affinity polymorphic formsof certain FcRs, an antibody or fusion polypeptide with a Fc regionoptimized for binding to one or more FcRs may result in more effectivedestruction of cancer cells.

In certain embodiments, modified Fc regions impart improved propertiesto a polypeptide or a complex which includes a polypeptide into whichthe Fc region is incorporated, e.g., a complex such as a full-lengthantibody, chimeric antibody or humanized antibody which includes an Igheavy chain having an modified Fc region, such as increased or modifiedbinding to one or more FcRs, and/or increased or modified antibodydependent cellular cytotoxicity (ADCC), as compared to a correspondingpolypeptide or complex, such as an antibody, incorporating acorresponding unmodified (a wild-type or parent) Fc region, or adifferent modified Fc region. In some embodiments of the invention,modified Fc regions impart increased or decreased half life to amolecule.

In one embodiment of the invention, a modified Fc region of theinvention contains one substitution. In other embodiments, a modified Fcregion of the invention contains two, three, four, five or moresubstitutions in combination, which may additively or synergisticallyenhance the properties of the modified Fc regions. In anotherembodiment, the invention includes a polypeptide having a modified Fcregion, i.e., it is an Fc fusion polypeptide that contains one of thesubstitutions. In one embodiment, the non-Fc region of the fusionpolypeptide includes a target binding molecule. In other embodiments,the invention includes a polypeptide having a modified Fc region of theinvention that contains two, three, four, five, six, ten, twelve, ormore substitutions in combination.

In addition to the polypeptide, protein or other complex, e.g., aconjugate, incorporating an modified Fc region, the invention alsoencompasses polynucleotides and expression vectors encoding a modifiedFc region or polypeptides having a modified Fc region, includinglibraries of those polynucleotides and expression vectors, host cellsinto which such polynucleotides or expression vectors have beenintroduced, for instance, so that the host cell produces a polypeptidehaving the modified Fc region, libraries of host cells, and methods ofmaking, culturing or manipulating the host cells or libraries of hostcells. For instance, the invention includes culturing such host cells sothat a polypeptide with a modified Fc region is produced, e.g., secretedor otherwise released from the host cell. Pharmaceutical compositionsand kits which include a polypeptide, protein or other complex with anmodified Fc region, and/or polynucleotides, expression vectors or hostcells encoding polypeptides having such a modified Fc region, are alsoencompassed. Moreover, use of a polypeptide, protein or conjugate withan modified Fc region, such as in Fc receptor binding assays or toinduce ADCC activity in vitro or in vivo, is also encompassed by theinvention. The invention also provides a polypeptide, protein,conjugate, polynucleotide, expression vector, and/or host cell of theinvention for use in medical therapy, as well as the use of apolypeptide, protein or other complex, polynucleotide, expressionvector, and/or host cell of the invention for the manufacture of amedicament, e.g., useful to induce ADCC activity in vitro or in vivo.

A “parent Fc”, as used herein, can be a naturally occurring Fc region ofan IgA, IgD, IgE, IgG or IgM class of antibody. Alternatively, thesource of a parent Fc is a Fc region from a naturally occurringantibody, including IgG1, IgG1, IgG3, IgG4, IgA1, or IgA2. A parent Fcregion to be modified may be selected for its FcR binding affinityand/or FcR binding pattern, and an modified Fc region has at least anenhanced affinity for at least one FcR, but may otherwise have the samepattern of FcR binding, as the parent Fc region.

A parent Fc region is preferably one that interacts with one or moreFcRs, including but not limited to FcγRs, FcαRs, FcμRs, FcδRs, FcRn, andviral FcγR. A modified Fc region derived from such a parent Fc region isone that has an enhanced interaction with one or more FcRs and enhancedADCC, relative to the parent Fc region. ADCC generally requires the Fcregion to be combined with a binding domain (e.g., an antibody variabledomain). Methods to detect FcR binding and ADCC are known to the art.

FcRs are defined by their specificity for immunoglobulin isotypes andare well known in the art.

An Fc containing fusion includes a polypeptide where a Fc region withfavorable FcR binding, and optionally favorable pharmacokinetics, islinked to one or more molecules. The linkage may be synthetic in nature,e.g., via chemical conjugation, or via recombinant expression, i.e., afusion polypeptide is formed. Thus, the molecule linked to a Fc regionmay be a molecule useful to isolate or purify the Fc region, e.g., a tagsuch as a Flag-tag, Strep-tag, glutathione S transferase, maltosebinding protein (MBP) or a His-tag, or other heterologous polypeptide,e.g., a ligand for a receptor, an extracellular domain of a receptor, ora variable region of a heavy Ig chain, and/or another molecule.

A vector encoding a modified Fc region or a Fc region containingpolypeptide such as an Ig heavy chain with a modified Fc region or otherFc fusion polypeptide may be introduced into a host cell, optionallyalong with other vectors, e.g., a vector encoding an Ig light chain, orinto a host cell modified to express another polypeptide such as an Iglight chain, or into an in vitro transcription/transcription reaction,so as to express the encoded polypeptide. The modified Fc region, Igheavy chain and Ig light chain may also be expressed in the same vectorand introduced into a host cell. For some expression systems, host cellsmay be cultured in conventional nutrient media modified as appropriatefor inducing promoters, selecting transformants, or amplifying desiredsequences. A resulting polypeptide with a modified Fc region isoptionally isolated, e.g., from host cell supernatants, and screened forone or more activities.

In one embodiment, the Fc region may be one that is anchored to thesurface of a cell, such as a host cell, e.g., via fusion with atransmembrane domain.

Suitable host cells for expressing the polynucleotide in the vectors arethe prokaryotic, yeast, or higher eukaryotic cells. Suitable prokaryotesfor this purpose include eubacteria, such as Gram-negative orGram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Kiebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis,Pseudomonas such as P. aeruginosa, and Streptomyces. Eukaryotic microbessuch as filamentous fungi or yeast are also suitable cloning orexpression hosts for polypeptide variant-encoding vectors. Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces hosts such as,e.g., K. lactis, K. fragilis, K. bulgaricus, K. wickeramii, K. waltii,K. drosophilarum, K. thermotolerans, and K. marxianus; Pichia pastoris,Candida, Trichoderma reesia, Schwanniomyces such as Schwanniomycesoccidentalis; and filamentous fungi such as Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts may be employed.

Suitable host cells for the expression of glycosylated polypeptides arederived from multicellular organisms. Examples of invertebrate cells forexpression of glycosylated polypeptide include plant and insect cells.Examples of eukaryotic cell generation, screening and production hostsinclude 3T3 mouse fibroblast cells, BHK21 Syrian hamster fibroblastcells, MDCK, dog epithelial cells, Hela human epithelial cells, PtK1 ratkangaroo epithelial cells, SP2/0 mouse plasma cells, and NS0 mouse mouseplasma cells, HEK 293 human embryonic kidney cells, COS monkey kidneycells, CHO, CHO-S Chinese hamster ovary cells, R1 mouse embryonic cells,E14.1 mouse embryonic cells, H1 human embryonic cells, H9 humanembryonic cells, PER C.6, and human embryonic cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda, Aedes aegypti,Aedes albopictus, Drosophila melanogaster, and Bombyx mori may be used.For instance, viral vectors maybe used to introduce a polynucleotide,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures of cotton, corn, potato, soybean, petunia, tomato, and tobaccocan also be utilized as hosts. Examples of useful vertebrate cellsinclude mammalian cells, e.g., human, simian, canine, feline, bovine,equine, caprine, ovine, swine, or rodent, e.g., rabbit, rat, mink ormouse cells, such as CHO cells. Transgenic plants and animals may beemployed as expression systems, although glycosylation patterns in thosecells may be different from human glycoproteins. In one embodiment,transgenic rodents are employed as expression systems. Bacterialexpression may also be employed. Although bacterially expressed proteinslack glycosylation, other alterations may compensate for any reducedactivity such as poor stability and solubility, which may result fromprokaryotic expression.

Optionally, an Fc region or Fc containing polypeptide is isolated fromhost cells, e.g., from host cell supernatants, or an in vitrotranscription/translation mixture, yielding a composition. An isolatedpolypeptide in the composition is one which has been isolated from atleast one other molecule found in host cells, host cell supernatants orthe transcription/translation mixture, e.g., by fractionation onimmunoaffinity or ion-exchange columns; ethanol precipitation; reversephase HPLC; chromatography on silica or on an anion-exchange resin suchas DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; or ligand affinitychromatography. For some applications, the isolated polypeptide in thecomposition is the predominant species present (i.e., on a molar basisit is more abundant than any other individual species in thecomposition), and preferably comprises at least about 50 percent (on amolar basis), more preferably more than about 85%, about 90%, about 95%,and about 99, of all macromolecular species present. The isolated Fcregion or Fc containing polypeptide may be subjected to further in vitroalterations, e.g., treated with enzymes or chemicals such as proteases,molecules such as those which alter glycosylation or ones that areuseful to conjugate (couple) the isolated Fc region or Fc regioncontaining polypeptide to another molecule such as a label including butnot limited to fluorescent labels (e.g., FITC, rhodamine, lanthanide,phosphors), enzymatic labels (e.g., horseradish peroxidase,/3-galactosidase, luciferase, alkaline phosphatase), chemiluminescentlabels, biotinyl groups, avidin groups, or polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags), sugars, lipids, fats, paramagnetic molecules or sound waveemitters, metals, or synthetic polymers.

Methods to screen for activities associated with polypeptides orcomplexes that incorporate a Fc region, including but not limited to FcRbinding (see, for example, U.S. Pat. Nos. 6,737,056, 7,217,797, and8,088,376, all incorporated herein by reference), are well known to theart. For instance, to assess ADCC activity of a Fc containingpolypeptide, an in vitro and/or in vivo ADCC assay, may be performedusing varying effector:target ratios, e.g., PBMC and NK cells or in aanimal model, respectively. In one embodiment, Fc containingpolypeptides expressed by host cells are screened for enhanced FcRreceptor binding affinity or activity in vitro and/or in vivo and/orADCC activity in vitro and/or in vivo. In one embodiment, the binding ofa FcR by a Fc containing polypeptide with an modified Fc region isgreater than the binding of that receptor by a corresponding polypeptidewith an unmodified Fc region.

Thus, by introducing amino acid sequence modifications described hereinin a wild-type or parent Fc region or a Fc region containingpolypeptide, which wild-type or parent Fc region preferably elicits ADCCand optionally is a human Fc region, e.g., a native sequence human Fcregion human IgG sequence, a modified Fc region is obtained which bindsFcR with better affinity and mediates ADCC in the presence of humaneffector cells more effectively than the wild-type or parent Fc regionor Fc region containing polypeptide. Soluble FcRs such as recombinantsoluble human CD16 and recombinant soluble human CD32 can be contactedwith one or more different modified Fc regions in parallel, and modifiedFc regions having one or more substitutions that enhance binding tohuman CD16 but not to human CD32, relative to an unmodified Fc region,are identified. Those substitutions may be combined with othersubstitutions that enhance binding. A combination of substitutions in anFc region or Fc region containing polypeptide may yield acombinatorially modified Fc region, or a combinatorially modified Fcregion containing polypeptide with synergistically enhanced properties.

Other methods to identify polypeptides with modified Fc regions,including antibodies with an modified Fc region, with desirableproperties, and thus a corresponding polynucleotide sequence, may beemployed alone or in combination with methods described above, includeusing modeling, e.g., 3D-modeling, of modified Fc regions, preferably inthe context of the molecule to be screened for activity, e.g., anantibody with the Fc region, to select for Fc regions with particularcharacteristics. Characteristics that may be screened for by modelinginclude, but are not limited to, a particular angle near FcR bindingsites, hinge architecture, and intra- and inter-molecular chaininteractions, e.g., substitutions that promote or disrupt hydrophobicinteractions or stabilize conformation in a particular region. Thus, a3D model of an Fc region containing polypeptide having at least one ormore substituted positions may be employed to identify combinations ofsubstitutions to be introduced into a polynucleotide for expression inhost cells.

The Fc variants, whether or not incorporated into a heterologouspolypeptide, e.g., incorporated into a Fc fusion with a ligand for acell surface receptor, e.g., CTLR-4 ligand or heavy chain of anantibody, or conjugated to a molecule of interest, as well aspolynucleotides and host cells encoding those variants, optionally incombination with one or more other agents, e.g., therapeutic or researchreagents, are useful in a variety of methods, e.g., in screeningmethods, prophylactic methods, therapeutic methods, veterinary methodsand agricultural methods. The one or more other agents include other Fcregion or Fc region containing polypeptides, including those withunmodified Fc regions. In one embodiment, an Fc variant is incorporatedinto an antibody or other Fc fusion polypeptide and that antibody or Fcfusion polypeptide, optionally in conjunction with one or more otheruseful compositions, is employed to target particular cells.

In one embodiment, an Fc variant containing antibody or anantigen-binding fragment thereof targets and optionally kills targetcells that bear the target antigen. In another embodiment, a Fc variantcontaining antibody or an antigen-binding fragment thereof targets andactivates cells that bear the target antigen, e.g., thereby increasingexpression of another antigen, such as a viral or cellular antigen. Inone embodiment, the Fc variants or polypeptides incorporating an Fcvariant may be used to prevent, inhibit or treat various conditions ordiseases, in humans and non-humans, including non-human mammals. Forexample, an antibody containing a modified Fc region may be administeredto a human or non-human animal which is at risk of, e.g., prone tohaving a disease, prior to the onset of the disease and so prevent orinhibit one or more symptoms of that disease. A Fc region or Fc regioncontaining polypeptide, or a conjugate thereof, may be administeredafter clinical manifestation of a disease in a human or non-human animalto inhibit or treat the disease. In one embodiment, a pharmaceuticalcomposition comprising an antibody or Fc fusion polypeptide isadministered to a human or non-human animal with an autoimmune,immunological, infectious, inflammatory, neurological, or neoplasticdisease, e.g., cancer.

Fc regions or a Fc region containing polypeptides may be administeredalone or in combination with one or more other therapeutic agents,including but not limited to cytotoxic agents, e.g., chemotherapeuticagents, cytokines, growth inhibitory agents, anti-hormonal agents,kinase inhibitors, anti-angiogenic agents, cardioprotectants, or othertherapeutic agents, in amounts that are effective for the purposeintended. The skilled medical practitioner can determine empirically theappropriate dose or doses of therapeutic agents including Fc regions orFc region containing polypeptides may thus be administered concomitantlywith one or more other therapeutic regimens. For example, an antibody orFc fusion polypeptide may be administered to a patient along withchemotherapy or other therapy, e.g., other agents such as ananti-angiogenic agent, a cytokine, radioisotope therapy, or bothchemotherapy and other therapies. In one embodiment, the antibody or Fcfusion may be administered in conjunction with one or more otherantibodies or Fc fusions, which may or may not comprise a Fc variant. Inone embodiment, a Fc containing polypeptide is administered with achemotherapeutic agent, i.e., a chemical compound useful in thetreatment of cancer. A chemotherapeutic or other cytotoxic agent may beadministered as a prodrug, i.e., it is in a form of a pharmaceuticallyactive substance that is less cytotoxic to cells compared to the drugand is capable of being converted into the drug.

Pharmaceutical compositions are also contemplated having an Fc region,an Fc fusion polypeptide, antibodies having an Fc region, or conjugatesthereof, optionally formulated with one or more other agents.Formulations of antibodies, Fc regions, or Fc region-containingpolypeptides, or conjugates are prepared for storage by mixing theantibodies, Fc regions, or Fc region-containing polypeptides, orconjugates, having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asantioxidants; alkyl parabens; low molecular weight (less than about 10residues) polypeptides; hydrophilic polymers; amino acids;monosaccharides; and other carbohydrates; chelating agents; fillers;binding agents; additives; coloring agents; salt-forming counter-ions;metal complexes; and/or non-ionic surfactants. Other formulationsinclude lipid or surfactant based formulations, and microparticle ornanoparticle based formulations, including sustained release dosageformulations, which are prepared by methods known in the art.

The concentration of the Fc region, antibody or other Fc regioncontaining polypeptide in the formulation may vary from about 0.1 to 100weight %. In a preferred embodiment, the concentration of the Fc region,antibody or Fc fusion polypeptide is in the range of 0.001 to 2.0 M. Inorder to treat a patient, an effective dose of the Fc region, orantibody or other Fc region-containing polypeptide, and conjugatesthereof, may be administered.

By “therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. Dosages may range from 0.01 to100 mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kgof body weight, with 1 to 30 mg/kg being preferred, although otherdosages may provide beneficial results. The amount administered isselected to prevent treat a particular condition or disease.Administration of the Fc region, or antibody or other Fcregion-containing polypeptide, and conjugates thereof, may be continuousor intermittent, depending, for example, upon the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of the Fc region, or antibody or otherFc region-containing polypeptide, and conjugates thereof, may beessentially continuous over a preselected period of time or may be in aseries of spaced doses. Both local and systemic administration iscontemplated.

Administration of the pharmaceutical composition comprising a Fc region,an antibody or other Fc containing polypeptide and conjugates,preferably in the form of a sterile aqueous solution, may be done in avariety of ways, including, but not limited to, orally, subcutaneously,intravenously, intranasally, intraotically, transdermally, topically,intraperitoneally, intramuscularly, intrapulmonary, inhalabletechnology, vaginally, parenterally, rectally, and intraocularly. Insome instances, for example for the treatment of wounds, inflammation,etc., the antibody or Fc fusion may be directly applied as a solution orspray.

Some references describing techniques that may be used in the evolvingstep of the present invention include Molecular Cloning: A LaboratoryManual (Sambrook et al, 3rd Ed., Cold Spring Harbor Laboratory Press,(2001); Harlow and Lane, Antibodies: A Laboratory Manual Cold SpringHarbor Laboratory Press, New York, 1988; Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed., United States Public HealthService, National Institutes of Health, Bethesda (1991); Carter et al.,Nucleic Acids Res., 13:4431 (1985) Kunkel et al., Proc. Natl. Acad. Sci.USA, 82:488 (1987); Higuchi, in PCR Protocols, ρρ. 177-183 (AcademicPress, 1990); Vallette et al., Nuc. Acids Res., 17:723 (1989) Wells etal., Gene, 34:315 (1985); Gazzano-Santoro et al., J. Immunol. Methods,202:163 (1996); Green et al., Nature Genet., 7:13 (1994); Lonberg etal., Nature, 368:856 (1994); Taylor et al., Int. Immun., 6:579 (1994);McCafferty et al., Nature, 348:552 (1990); Johnson and Chiswell, CurrentOpinion in Structural Biology, 3 :5564 (1993); Dall'Acqua, et al., TheJournal of Immunology, 169: 5171-5180 (2002); Yeung, et al., The Journalof Immunology, 182: 7663-7671 (2009); Zalevsky, et al., NatureBiotechnology; doi: 10.1038/nbt.1601 (published online 17 Jan. 2010);and Dall'Acqua, et al., The Journal of Biological Chemistry, Vol 281,Num 33, 23515-23524 (2006), the disclosures of which are herebyincorporated by reference in their entirety.

Expression of Evolved Molecules

Once a library of mutant molecules is generated, DNA can be expressedusing routine molecular biology techniques. Thus, protein expression canbe directed using various known methods.

For example, briefly, a wild type gene can be evolved using any varietyof random or non-random methods such as those indicated herein. MutantDNA molecules are then digested and ligated into vector DNA, such asplasmid DNA using standard molecular biology techniques. Vector DNAcontaining individual mutants is transformed into bacteria or othercells using standard protocols. This can be done in an individual wellof a multi-well tray, such as a 96-well tray for high throughputexpression and screening. The process is repeated for each mutantmolecule.

Polynucleotides selected and isolated as described are introduced into asuitable host cell. A suitable host cell is any cell which is capable ofpromoting recombination and/or reductive reassortment. The selectedpolynucleotides are preferably already in a vector which includesappropriate control sequences. The host cell can be a higher eukaryoticcell, such as a mammalian cell, or a lower eukaryotic cell, such as ayeast cell, or preferably, the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (e.g. Ecker and Davis, 1986, Inhibitionof gene expression in plant cells by expression of antisense RNA, ProcNatl Acad Sci USA, 83:5372-5376).

As representative examples of expression vectors which may be used,there may be mentioned viral particles, baculovirus, phage, plasmids,phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA(e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, aspergillus and yeast).Thus, for example, the DNA may be included in any one of a variety ofexpression vectors for expressing a polypeptide. Such vectors includechromosomal, nonchromosomal and synthetic DNA sequences. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. The following vectors are provided by way ofexample; Bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNHvectors, (lambda-ZAP vectors (Stratagene); ptrc99a, ρKK223-3, pDR540,pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV,pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vectormay be used so long as they are replicable and viable in the host. Lowcopy number or high copy number vectors may be employed with the presentdisclosure.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lad, lacZ, T3,T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus, and mouse metallothionein-1. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using chloramphenicoltransferase (CAT) vectors or other vectors with selectable markers. Inaddition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

Therefore, in another aspect of the disclosure, novel polynucleotidescan be generated by the process of reductive reassortment. The methodinvolves the generation of constructs containing consecutive sequences(original encoding sequences), their insertion into an appropriatevector, and their subsequent introduction into an appropriate host cell.The reassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences, and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals, and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

In one aspect, the host organism or cell comprises a gram negativebacterium, a gram positive bacterium or a eukaryotic organism. Inanother aspect of the disclosure, the gram negative bacterium comprisesEscherichia coli, or Pseudomonas fluorescens. In another aspect of thedisclosure, the gram positive bacterium comprise Streptomyces diversa,Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, orBacillus subtilis. In another aspect of the disclosure, the eukaryoticorganism comprises Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, Kluyveromyces lactis, Hansenula plymorpha, orAspergillus niger. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Streptomyces,Salmonella typhimurium; fungal cells, such as yeast; insect cells suchas Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS orBowes melanoma; adenoviruses; and plant cells. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

With particular references to various mammalian cell culture systemsthat can be employed to express recombinant protein, examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described in “SV40-transformed simian cells support thereplication of early SV40 mutants” (Gluzman, 1981), and other cell linescapable of expressing a compatible vector, for example, the C127, 3T3,CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprisean origin of replication, a suitable promoter and enhancer, and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The cells are then propagated and “reductive reassortment” is effected.The rate of the reductive reassortment process may be stimulated by theintroduction of DNA damage if desired, in vivo reassortment is focusedon “inter-molecular” processes collectively referred to as“recombination” which in bacteria, is generally viewed as a“RecA-dependent” phenomenon. The disclosure can rely on recombinationprocesses of a host cell to recombine and re-assort sequences, or thecells' ability to mediate reductive processes to decrease the complexityof quasi-repeated sequences in the cell by deletion. This process of“reductive reassortment” occurs by an “intra-molecular”,RecA-independent process. The end result is a reassortment of themolecules into all possible combinations.

Host cells containing the polynucleotides of interest can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

Protein expression can be induced by a variety of known methods, andmany genetic systems have been published for induction of proteinexpression. For example, with appropriate systems, the addition of aninducing agent will induce protein expression. Cells are then pelletedby centrifugation and the supernatant removed. Periplasmic protein canbe enriched by incubating the cells with DNAse, RNAse, and lysozyme.After centrifugation, the supernatant, containing the new protein, istransferred to a new multi-well tray and stored prior to assay.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract is retained forfurther purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

The clones which are identified as having the desired activity may thenbe sequenced to identify the polynucleotide sequence encoding an enzymehaving the enhanced activity.

The polypeptides that are identified from such libraries can be used fortherapeutic, diagnostic, research and related purposes, and/or can besubjected to one or more additional cycles of shuffling and/orselection. The disclosure provides for a fragment of the conditionallyactive biologic protein which is at least 10 amino acids in length, andwherein the fragment has activity.

The disclosure provides for a codon-optimized polypeptide or a fragmentthereof, having enzyme activity, wherein the codon usage is optimizedfor a particular organism or cell. Narum et al., “Codon optimization ofgene fragments encoding Plasmodium falciparum merzoite proteins enhancesDNA vaccine protein expression and immunogenicity in mice”. Infect.Immun. 2001 December, 69(12):7250-3 describes codon-optimization in themouse system. Outchkourov et al., “Optimization of the expression ofEquistatin in Pichia pastoris, protein expression and purification”,Protein Expr. Purif 2002 February; 24(1): 18-24 describescodon-optimization in the yeast system. Feng et al., “High levelexpression and mutagenesis of recombinant human phosphatidylcholinetransfer protein using a synthetic gene: evidence for a C-terminalmembrane binding domain” Biochemistry 2000 Dec. 19, 39(50): 15399-409describes codon-optimization in E. coli. Humphreys et al., “High-levelperiplasmic expression in Escherichia coli using a eukaryotic signalpeptide: importance of codon usage at the 5′ end of the codingsequence”, Protein Expr. Purif. 2000 Nov. 20(2):252-64 describes howcodon usage affects secretion in E. coli.

The evolution of a conditionally active biologic protein can be aided bythe availability of a convenient high throughput screening or selectionprocess.

Once identified, polypeptides and peptides of the disclosure can besynthetic, or be recombinantly generated polypeptides. Peptides andproteins can be recombinantly expressed in vitro or in vivo. Thepeptides and polypeptides of the disclosure can be made and isolatedusing any method known in the art. Polypeptide and peptides of thedisclosure can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) “New chemicalmethods for synthesizing polynucleotides”, Nucleic Acids Res. Symp. Ser.215-223; Horn (1980), “Synthesis of oligonucleotides on cellulose. PartII: design and synthetic strategy to the synthesis of 22oligodeoxynucleotides coding for Gastric Inhibitory Polypeptide (GIP)”,Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K., TherapeuticPeptides and Proteins, Formulation, Processing and Delivery Systems(1995) Technomic Publishing Co., Lancaster, Pa. For example, peptidesynthesis can be performed using various solid-phase techniques (seee.g., Roberge (1995) “A strategy for a convergent synthesis of N-linkedglycopeptides on a solid support”, Science 269:202; Merrifield (1997)“Concept and early development of solid-phase peptide synthesis”,Methods Enzymol. 289:3-13) and automated synthesis may be achieved,e.g., using the ABI 43 IA Peptide Synthesizer (Perkin Elmer) inaccordance with the instructions provided by the manufacturer.

The peptides and polypeptides of the disclosure can also beglycosylated. The glycosylation can be added post-translationally eitherchemically or by cellular biosynthetic mechanisms, wherein the latterincorporates the use of known glycosylation motifs, which can be nativeto the sequence or can be added as a peptide or added in the nucleicacid coding sequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the disclosure, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the disclosure. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the disclosure which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the disclosure, i.e., that its structureand/or function is not substantially altered.

Polypeptide mimetic compositions of the disclosure can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the disclosure include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the disclosure can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,˜C(.dbd.O)˜CH.sub.2˜ for —C(.dbd.O)˜NH—), aminomethylene (CH.sub.2-NH),ethylene, olefin (CH.dbd.CH), ether (CH.sub.2˜O), thioether(CH.sub.2˜S), tetrazole (CN.sub.4-), thiazole, retroamide, thioamide, orester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of AminoAcids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide BackboneModifications,” Marcell Dekker, N. Y.).

A polypeptide of the disclosure can also be characterized as a mimeticby containing all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1,-2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylanines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′˜N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-moφholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the disclosure canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S-form.

The mimetic polypeptides of the present invention may be synthesizedusing any protein chemical synthesis techniques. In a typical in vitroprotein synthesis process, a peptide is extended in length by one aminoacid through forming a peptide bond between the peptide and an aminoacid. The formation of the peptide bond is carried out using a ligationreaction, which can use a natural amino acid or a non-natural aminoacid. Thus, in this manner non-natural amino acids can be introducedinto the polypeptides of the present invention to make mimetics.

The conditionally active biologic proteins can also be synthesized, as awhole or in part, using chemical protein synthesis methods well known inthe art. See e.g., Caruthers (1980) “New chemical methods forsynthesizing polynucleotides”, Nucleic Acids Res. Symp. Ser. 215-223;Horn (1980), “Synthesis of oligonucleotides on cellulose. Part II:design and synthetic strategy to the synthesis of 22oligodeoxynucleotides coding for Gastric Inhibitory Polypeptide (GIP)¹”, Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K., TherapeuticPeptides and Proteins, Formulation, Processing and Delivery Systems(1995) Technomic Publishing Co., Lancaster, Pa. For example, peptidesynthesis can be performed using various solid-phase techniques (seee.g., Roberge (1995) “A strategy for a convergent synthesis of N-linkedglycopeptides on a solid support”, Science 269:202; Merrifield (1997)“Concept and early development of solid-phase peptide synthesis”,Methods Enzymol. 289:3-13) and automated synthesis may be achieved,e.g., using the ABI 43 IA Peptide Synthesizer (Perkin Elmer) inaccordance with the instructions provided by the manufacturer.

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments thereof. Such methods have beenknown in the art since the early 1960's (Merrifield, R. B., “Solid-phasesynthesis.I. The synthesis of a tetrapeptide”, J. Am. Chem. Soc,85:2149-2154, 1963) (See also Stewart, J. M. and Young, J. D., SolidPhase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill.,pp. 11-12)) and have recently been employed in commercially availablelaboratory peptide design and synthesis kits (Cambridge ResearchBiochemicals). Such commercially available laboratory kits havegenerally utilized the teachings of H. M. Geysen et al., “Use of peptidesynthesis to probe viral antigens for epitopes to a resolution of asingle amino acid,” Proc. Natl. Acad. Sci., USA, 81:3998 (1984) andprovide for synthesizing peptides upon the tips of a multitude of “rods”or “pins” all of which are connected to a single plate.

The mimetic polypeptides of the present invention may also be producedby recombinant techniques, which produce a polypeptide by inserting acoding sequence of the polypeptide into an expression vector andutilizing the protein translation machinery of a eukaryotic cellproduction host. The protein translation machinery reads the codons ofthe coding sequence and uses tRNA to bring in the encoded amino acid toproduce the polypeptide. There are several techniques can be used toalter the protein translation machinery to allow it to incorporate anon-natural amino acid into a recombinant polypeptide. A proven approachdepends on the recognition of the non-natural amino acid byaminoacyl-tRNA synthetases, which, in general, require high selectivityto insure the fidelity of protein translation. These synthetases may beengineered to relax the substrate specificity such that a non-naturalamino acid may be linked to a tRNA, which then brings the non-naturalamino acid to the protein translation machinery to be incorporated intoa polypeptide. For example, it was found that replacement of Ala²⁹⁴byGly in Escherichia coli phenylalanyl-tRNA synthetase (PheRS) increasesthe size of substrate binding pocket, and results in the acylation oftRNA^(Phe) by p-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast andH. Hennecke, Biochemistry, 33:7107 (1994). An Escherichia coli strainharboring this mutant PheRS allows the incorporation ofp-Cl-phenylalanine or p-Br-phenylalanine in place of phenylalanine, See,e.g., M. Ibba and H. Hennecke, FEBS Lett., 364:272 (1995); and, N.Sharma, R. Furter, P. Kast and D. A. Tirrell, FEBS Lett., 467:37 (2000).Similarly, a point mutation Phe130Ser near the amino acid binding siteof Escherichia coli tyrosyl-tRNA synthetase was shown to allowazatyrosine to be incorporated more efficiently than tyrosine. See, F.Hamano-Takaku, T. Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M.Kitabatake, D. Soll and S. Nishimura, J. Biol. Chem., 275:40324 (2000).

The first method involves reassigning sense codon, which engineers atleast one aminoacyl-tRNA synthetase. The enzyme normally adds a naturalamino acid to a tRNA to be transported to the protein translationmachinery for protein synthesis. However, an aminoacyl-tRNA synthetasefor a particular tRNA may be altered such that it can have certain levelof promiscuity to charge a non-natural amino acid non-specifically tothe tRNA to activate the tRNA. The activated tRNA can carry thenon-natural amino acid to the protein translation machinery (e.g.ribosomes) and add the non-natural amino acid to a peptide where a codonof the coding sequence calls for that particular tRNA. In other words,the codon for that particular tRNA has been reassigned to non-naturalamino acids. The recombinant polypeptide comprises 19 natural aminoacids and at least one non-natural amino acid. The one natural aminoacid in the polypeptide has been replaced by at least one non-naturalamino acid. The successful substitution of a natural amino acid withnon-natural amino acid relies on the use of auxotrophic expression hostsdeficient in the biosynthesis of that natural amino acid. Employment ofsuch hosts limits competition from the natural amino acid for thereassigned sense codon, and improves the incorporation efficiency andyield of target proteins. Codons for many amino acids (including Met,Pro, Tyr, Phe, Leu, Val etc.) have been reassigned, and more than 60non-natural amino acids have been incorporated into proteins via thismethod. See Hendrickson et al., “Incorporation of nonnatural amino acidsinto proteins,” Annu. Rev. Biochem., vol. 73, pages 147-176, 2004;Voloshchuk et al., “Incorporation of unnatural amino acids for syntheticbiology,” Mol. Biosyst., vol. 6, pages 65-80, 2010, both incorporatedherein by reference.

The main limitation of this method is that the non-natural amino acidwill replace the natural amino acid throughout the polypeptide sequence,which may restrict its application if such global substitution isundesirable. One solution is to mutate sites where substitution isundesirable to other natural amino acids so that only the desiredsite(s) are reserved for the non-natural amino acid. With thismodification, the method can introduce a non-natural amino acidsite-specifically at any desired site to produce mimetic polypeptides.

Another method for producing mimetic recombinant polypeptides is byusing wobble codons. Wobble codons refer to codons that are decoded bytRNAs via non-classical Watson-Crick base-pairing. The non-classical (orwobble) pairing is enabled through modification at the tRNA's 1^(st)anticodon base (which pairs with the 3rd base to the codon triplet), asproposed in the “Wobble Hypothesis”. For example, many organisms haveonly one tRNA to decode two codons for Phe: UUU and UUC. As a result,the GAA anticodon on the tRNA binds to the UUC codon via Watson-Crickbase-pairing, and to the UUU codon via “wobble” base-pairing.

Because of the wobble pairing between codon and anticodon, one tRNA maypair with several codons, and a given codon may pair with more than onetRNA. Taking advantage of this property, a wobble codon may be assignedto a non-natural amino acid to generate a recombinant protein thatcontains natural amino acids and at least one non-natural amino acid.For example, Phe is normally encoded by two codons UUC and UUU, withboth codons recognized by a single tRNA. By expressing an orthogonalpair of aminoacyl-tRNA synthetase and tRNA, with specificity for anon-natural amino acid and containing the “AAA” anticodon, efficientintroduction of the non-natural amino acid at UUU codons can be achieved(Kwon et al., “Breaking the degeneracy of the genetic code,” J. Am.Chem. Soc., vol. 125, pages 7512-7513, 2003, incorporated herein byreference). With this method, Phe can be essentially quantitativelyassigned to the UUC codon, and a non-natural amino acid to the UUUwobble codon. Furthermore, multiple copies of a non-natural amino acidcan be introduced site specifically into a polypeptide.

The third method for generating recombinant mimetic polypeptide is byusing biased codons. The preferred codons differ between organisms, andeven between different tissues or cell types of the same organism. Thecellular content of tRNA species is a determining factor on the ratesand amounts of protein synthesized. As a consequence, recombinantprotein production in heterologous host cells is often codon-optimizedto match the preferred host cell codon bias (The codon usage databasefor different organisms and codon analysis of a given gene can be foundat: http://www.kazusa.or.jp/codon/).

The biased codon usage provides another method to introduce non-naturalamino acids into recombinant polypeptides. For example, out of the sixdegenerate codons for Arg, AGG and AGA are rarely used in E. coli.Introduction of an orthogonal pair of aminoacyl-tRNA synthetase and tRNAthat pairs with the AGG codon into an E. coli expression host may enablelinking a non-natural amino acid to the tRNA. Therefore, the tRNA with anon-natural amino acid linked thereto can bring the non-natural aminoacid to the codon AGG, where normally Arg may be encoded. This methodhas been proven feasible with an in vitro cell-free biased system, wherechemically synthesized non-natural amino acid linked tRNA that pairswith the AGG codon was incorporated at AGG codons (Hohsaka et al., FEBSLetters, vol. 344, pages 171-174, 1994). The method could be adapted toan E. coli cell-based expression system if an aminoacyl-tRNA synthetasecan be engineered to link a non-natural orthogonal to a tRNA.

Similarly, a bias codon may be assigned to a non-natural amino acid inmammalian cells that exhibit codon bias. For example, through study ofhuman papillomavirus gene expression in different mammalian cells,Frazer and his colleagues have found that papillomavirus proteinexpression is determined by the codon usage and tRNA availability.Substantial differences in the tRNA pools were discovered betweendifferentiated and undifferentiated keratinocytes (Zhao et al., “Genecodon composition determines differentiation-dependent expression of aviral capsid gene in keratinocytes in vitro and in vivo,” Mol. CellBiol., vol. 25, pages 8643-8655, 2005), and the observed bias in theirtRNA may be the reason that papillomavirus replicates exclusively inepithelial cells. For example, in CHO and Cos1 cells, it seems that TCGis a bias and thus might be assigned to a non-natural amino acid.

As the codon bias phenomenon is wide-spread in different eukaryoticorganisms, utilization of such codons for site-specific incorporation ofnon-natural amino acids could be applied in many eukaryotic cellproduction hosts. The limitation would be the engineering of theaminoacyl-tRNA synthetase to link a non-natural orthogonal to a tRNAthat can pair with a biased codon in the production hosts.

A fourth method for producing a mimetic polypeptide is by suppressing astop codon. Generally, protein translation terminates at one of thethree stop codons (encoded by UAG (amber), UAA (ochre) and UGA (opal))by the action of protein release factors (RF). However, occasionalread-through of a stop codon with an amino acid has been observed tohappen naturally in a variety of species. The suppression is caused byeither mutations in the tRNA anticodon or mismatches of thecodon-anticodon (Beier & Grimm, “Misreading of termination codons ineukaryotes by natural nonsense suppressor tRNAs,” Nucleic Acids Res.,vol. 29, pages 4767-4782, 2001). Utilization of stop codon suppressionrepresents another way to producing proteins containing non-naturalamino acids, and generally involves the introduction of anaminoacyl-tRNA synthetase that can link a non-natural amino acid to atRNA that can pair with a stop codon. For example, an aminoacyl-tRNAsynthetase and tRNA that pairs with the amber stop codon has beendeveloped to introduce a non-natural amino acid site-specifically atamber codons, as it is the least frequently used stop codon in botheukaryotic (23% in humans) and prokaryotic genomes (7% in E. coli) (Liuet al., “Genetic incorporation of unnatural amino acids into proteins inmammalian cells,” Nat. Methods, vol. 4, pages 239-244, 2007). Ochre andopal stop codons have been used for the introduction of non-naturalamino acids as well (Kohrer et al., “Complete set of orthogonal 21^(St)aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs:concomitant suppression of three different termination codons in an mRNAin mammalian cells,” Nucleic Acids Res., vol. 32, pages 6200-6211,2004). So far, over 70 non-natural amino acids have beensite-specifically incorporated into recombinant proteins by this method(Liu & Schultz, “Adding new chemistries to the genetic code,” Annu. Rev.Biochem., vol. 79, pages 413-444, 2010). Typically, over 95% non-naturalamino acid incorporation efficiency (defined as occupancy rate ofnon-natural amino acid in the full-length product) at the desired sitecan be obtained, making it one of the most frequently used methods fornon-natural amino acid incorporation.

The present invention also encompasses any other techniques known to aperson skilled in the art for introducing non-natural amino acids into arecombinant polypeptide. Some of the techniques involve usingfour-base-pair codons (Anderson et al., “An expanded genetic code with afunctional quadruplet codon. Proc. Natl. Acad. Sci. U.S.A., vol. 101,pages 7566-7571, 2004). More discussion about producing mimeticrecombinant polypeptides may be found in U.S. Pat. No. 7,045,337 andWO2010132341A2, both of which are hereby incorporated herein byreference.

The disclosure also provides methods for modifying the polypeptides ofthe disclosure by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques. Modifications can occur anywhere in thepolypeptide, including the peptide backbone, the amino acid side-chainsand the amino or carboxyl termini. It will be appreciated that the sametype of modification may be present in the same or varying degrees atseveral sites in a given polypeptide. Also a given polypeptide may havemany types of modifications. Modifications include acetylation,acylation, PEGylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of a phosphatidylinositol,cross-linking cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristolyation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,and transfer-RNA mediated addition of amino acids to protein such asarginylation. See, e.g., Creighton, T. E., Proteins—Structure andMolecular Properties 2nd Ed., W. H. Freeman and Company, New York(1993); Posttranslational Covalent Modification of Proteins, B. C.Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the disclosure. Such methodshave been known in the art since the early 1960's (Merrifield, R. B.,“Solid-phase synthesis.I. The synthesis of a tetrapeptide”, J. Am. Chem.Soc, 85:2149-2154, 1963) (See also Stewart, J. M. and Young, J. D.,Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford,Ill., pp. 11-12)) and have recently been employed in commerciallyavailable laboratory peptide design and synthesis kits (CambridgeResearch Biochemicals). Such commercially available laboratory kits havegenerally utilized the teachings of H. M. Geysen et al., “Use of peptidesynthesis to probe viral antigens for epitopes to a resolution of asingle amino acid,” Proc. Natl. Acad. Sci., USA, 81:3998 (1984) andprovide for synthesizing peptides upon the tips of a multitude of “rods”or “pins” all of which are connected to a single plate. When such asystem is utilized, a plate of rods or pins is inverted and insertedinto a second plate of corresponding wells or reservoirs, which containsolutions for attaching or anchoring an appropriate amino acid to thepin's or rod's tips. By repeating such a process step, i.e., invertingand inserting the rod's and pin's tips into appropriate solutions, aminoacids are built into desired peptides. In addition, a number ofavailable FMOC peptide synthesis systems are available. For example,assembly of a polypeptide or fragment can be carried out on a solidsupport using an Applied Biosystems, Inc. Model 431 A™ automated peptidesynthesizer. Such equipment provides ready access to the peptides of thedisclosure, either by direct synthesis or by synthesis of a series offragments that can be coupled using other known techniques.

The synthetic polypeptide or fragment thereof can be recovered andpurified by known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the polypeptide. If desired, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The disclosure provides for a conditionally active protein variantpreparation or formulation which comprises at least one of the proteinvariants, wherein the preparation is liquid or dry. The proteinformulation optionally includes a buffer, cofactor, second or additionalprotein, or one or more excipients. In one aspect the formulation isutilized as a therapeutic conditionally active biologic protein which isactive under aberrant or non-physiological conditions, but less activeor inactive under normal physiological conditions of, e.g., temperature,pH, or osmotic pressure, oxidation or osmolality.

Standard purification techniques can be employed for either recombinantor synthetic conditionally active biologic proteins.

Screening of Mutants to Identify Reversible or Nonreversible Mutants

Identifying desirable molecules is most directly accomplished bymeasuring protein activity at the permissive condition and the wild typecondition. The mutants with the largest ratio of activity(permissive/wild type) can then be selected and permutations of thepoint mutations are generated by combining the individual mutationsusing standard methods. The combined permutation protein library is thenscreened for those proteins displaying the largest differential activitybetween the permissive and wild type condition.

Activity of supernatants can be screened using a variety of methods, forexample using high throughput activity assays, such as fluorescenceassays, to identify protein mutants that are sensitive at whatevercharacteristic one desires (temperature, pH, etc). For example, toscreen for temporally sensitive mutants, the enzymatic or antibodyactivity of each individual mutant is determined at lower temperatures(such as 25 degrees Celsius), and at temperatures which the originalprotein functions (such as 37 degrees Celsius), using commerciallyavailable substrates. Reactions can initially be performed in a multiwell assay format, such as a 96-well assay, and confirmed using adifferent format, such as a 14 ml tube format.

The disclosure further provides a screening assay for identifying aenzyme, the assay comprising: (a) providing a plurality of nucleic acidsor polypeptides; (b) obtaining polypeptide candidates to be tested forenzyme activity from the plurality; (c) testing the candidates forenzyme activity; and (d) identifying those polypeptide candidates whichexhibit elevated enzyme activity under aberrant or non-physiologicalconditions, and decreased enzyme activity compared to the wild-typeenzyme protein under normal physiological conditions of, e.g.,temperature, pH, oxidation, osmolality, electrolyte concentration orosmotic pressure.

In one aspect, the method further comprises modifying at least one ofthe nucleic acids or polypeptides prior to testing the candidates forconditional biologic activity, in another aspect, the testing of step(c) further comprises testing for improved expression of the polypeptidein a host cell or host organism, in a further aspect, the testing ofstep (c) further comprises testing for enzyme activity within a pH rangefrom about pH 3 to about pH 12. In a further aspect, the testing of step(c) further comprises testing for enzyme activity within a pH range fromabout pH 5 to about pH 10. In a further aspect, the testing of step (c)further comprises testing for enzyme activity within a pH range fromabout pH 6 to about pH 8. In a further aspect, the testing of step (c)further comprises testing for enzyme activity at pH 6.7 and pH 7.5. Inanother aspect, the testing of step (c) further comprises testing forenzyme activity within a temperature range from about 4 degrees C. toabout 55 degrees C. In another aspect, the testing of step (c) furthercomprises testing for enzyme activity within a temperature range fromabout 15 degrees C. to about 47 degrees C. In another aspect, thetesting of step (c) further comprises testing for enzyme activity withina temperature range from about 20 degrees C. to about 40 degrees C. Inanother aspect, the testing of step (c) further comprises testing forenzyme activity at the temperatures of 25 degrees C. and 37 degrees C.In another aspect, the testing of step (c) further comprises testing forenzyme activity under normal osmotic pressure, and aberrant (positive ornegative) osmotic pressure, In another aspect, the testing of step (c)further comprises testing for enzyme activity under normal electrolyteconcentration, and aberrant (positive or negative) electrolyteconcentration. The electrolyte concentration to be tested is selectedfrom one of calcium, sodium, potassium, magnesium, chloride, bicarbonateand phosphate concentration, in another aspect, the testing of step (c)further comprises testing for enzyme activity which results in astabilized reaction product.

In another aspect, the disclosure provides for a purified antibody thatspecifically binds to the polypeptide of the disclosure or a fragmentthereof, having enzyme activity, In one aspect, the disclosure providesfor a fragment of the antibody that specifically binds to a polypeptidehaving enzyme activity.

Antibodies and Antibody-Based Screening Methods

The disclosure provides isolated or recombinant antibodies thatspecifically bind to an antigen of the disclosure. These antibodies canbe used to isolate, identify or quantify the antigens of the disclosureor related polypeptides. These antibodies can be used to isolate otherpolypeptides within the scope the disclosure or other related proteins.The antibodies can be designed to bind to an active site of an enzyme.Thus, the disclosure provides methods of inhibiting enzymes using theantibodies of the disclosure.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the disclosure.Alternatively, the methods of the disclosure can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the disclosure.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)“Continuous cultures of fused cells secreting antibody of predefinedspecificity”, Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORYMANUAL, Cold Spring Harbor Publications, New York. Antibodies also canbe generated in vitro, e.g., using recombinant antibody binding siteexpressing phage display libraries, in addition to the traditional invivo methods using animals. See, e.g., Hoogenboom (1997) “Designing andoptimizing library selection strategies for generating high-affinityantibodies”, Trends Biotechnol. 15:62-70; and Katz (1997) “Structuraland mechanistic determinants of affinity and specificity of ligandsdiscovered or engineered by phage display”, Annu. Rev. Biophys. Biomol.Struct. 26:27-45.

Polypeptides or peptides can be used to generate antibodies which bindspecifically to the polypeptides, e.g., the enzymes, of the disclosure.The resulting antibodies may be used in immunoaffinity chromatographyprocedures to isolate or purify the polypeptide or to determine whetherthe polypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides of the disclosure.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of thedisclosure. After a wash to remove non-specifically bound proteins, thespecifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of thedisclosure can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to a non-human animal.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the disclosure. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof. Antibodies generated against thepolypeptides of the disclosure may be used in screening for similarpolypeptides (e.g., enzymes) from other organisms and samples. In suchtechniques, polypeptides from the organism are contacted with theantibody and those polypeptides which specifically bind the antibody aredetected. Any of the procedures described above may be used to detectantibody binding.

Screening Methodologies and “On-line” Monitoring Devices

In practicing the methods of the disclosure, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the disclosure, e.g., to screen polypeptides for enzymeactivity, to screen compounds as potential modulators, e.g., activatorsor inhibitors, of an enzyme activity, for antibodies that bind to apolypeptide of the disclosure, for nucleic acids that hybridize to anucleic acid of the disclosure, to screen for cells expressing apolypeptide of the disclosure and the like.

Arrays or “Biochips”

Nucleic acids or polypeptides of the disclosure can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the disclosure. For example, in oneaspect of the disclosure, a monitored parameter is transcript expressionof an enzyme gene. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the disclosure. Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins. The present disclosure can be practiced with any known“array,” also referred to as a “microarray” or “nucleic acid array” or“polypeptide array” or “antibody array” or “biochip,” or variationthereof. Arrays are generically a plurality of “spots” or “targetelements,” each target element comprising a defined amount of one ormore biological molecules, e.g., oligonucleotides, immobilized onto adefined area of a substrate surface for specific binding to a samplemolecule, e.g., mRNA transcripts.

In practicing the methods of the disclosure, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) “Gene chips: Array of hope for understanding generegulation”, Curr. Biol. 8:R171-R174; Schummer (1997) “InexpensiveHandheld Device for the Construction of High-Density Nucleic AcidArrays”, Biotechniques 23:1087-1092; Kern (1997) “Direct hybridizationof large-insert genomic clones on high-density gridded cDNA filterarrays”, Biotechniques 23:120-124; Solinas-Toldo (1997) “Matrix-BasedComparative Genomic Hybridization: Biochips to Screen for GenomicImbalances”, Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999)“Options Available—From Start to Finish—for Obtaining Expression Data byMicroarray”, Nature Genetics Supp. 21:25-32. See also published U.S.patent applications Nos. 20010018642; 20010019827; 20010016322;20010014449; 20010014448; 20010012537; 20010008765.

Capillary Arrays

Capillary arrays, such as the GIGAMATRIX™ Diversa Corporation, SanDiego, Calif., can be used in the methods of the disclosure. Nucleicacids or polypeptides of the disclosure can be immobilized to or appliedto an array, including capillary arrays. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of thedisclosure. Capillary arrays provide another system for holding andscreening samples. For example, a sample screening apparatus can includea plurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The apparatus can further include interstitialmaterial disposed between adjacent capillaries in the array, and one ormore reference indicia formed within of the interstitial material. Acapillary for screening a sample, wherein the capillary is adapted forbeing bound in an array of capillaries, can include a first walldefining a lumen for retaining the sample, and a second wall formed of afiltering material, for filtering excitation energy provided to thelumen to excite the sample. A polypeptide or nucleic acid, e.g., aligand, can be introduced into a first component into at least a portionof a capillary of a capillary array. Each capillary of the capillaryarray can comprise at least one wall defining a lumen for retaining thefirst component. An air bubble can be introduced into the capillarybehind the first component. A second component can be introduced intothe capillary, wherein the second component is separated from the firstcomponent by the air bubble. A sample of interest can be introduced as afirst liquid labeled with a detectable particle into a capillary of acapillary array, wherein each capillary of the capillary array comprisesat least one wall defining a lumen for retaining the first liquid andthe detectable particle, and wherein the at least one wall is coatedwith a binding material for binding the detectable particle to the atleast one wall. The method can further include removing the first liquidfrom the capillary tube, wherein the bound detectable particle ismaintained within the capillary, and introducing a second liquid intothe capillary tube. The capillary array can include a plurality ofindividual capillaries comprising at least one outer wall defining alumen. The outer wall of the capillary can be one or more walls fusedtogether. Similarly, the wall can define a lumen that is cylindrical,square, hexagonal or any other geometric shape so long as the walls forma lumen for retention of a liquid or sample. The capillaries of thecapillary array can be held together in close proximity to form a planarstructure. The capillaries can be bound together, by being fused (e.g.,where the capillaries are made of glass), glued, bonded, or clampedside-by-side. The capillary array can be formed of any number ofindividual capillaries, for example, a range from 100 to 4,000,000capillaries. A capillary array can form a micro titer plate having about100,000 or more individual capillaries bound together.

Engineering Conditionally Active Biological Proteins

The conditionally active biological proteins of the present invention,including the conditionally active antibodies against BBB-R andconditionally active antibodies for synovial fluid, tumormicroenvironments and stem cell niches, including cancer stem cells, maybe engineered by one or more protein engineering techniques describedherein. Non-limiting examples of protein engineering techniques alsoinclude antibody conjugation, engineering multispecific antibodies,engineering Fc region of the antibodies.

Conjugating Conditionally Active Biological Proteins

The conditionally active biological proteins provided by the presentinvention may be conjugated to a molecule. Because the conditionallyactive biological protein preferentially acts in the brain, synovialfluid, a tumor microenvironment, or a stem cell niche, the conditionallyactive biological protein may be conjugated to a molecule (therapeuticor diagnostic agent), which will be transported to the brain, synovialfluid, tumor microenvironment or stem cell niche with the conditionallyactive biological proteins. In some embodiments, the molecule hasnon-specific toxicity, which may be reduced by being conjugated to theconditionally active biological proteins, to thus preferentially act onthe disease site.

In some embodiments, the conjugated molecule on the conditionally activebiological protein may be optionally released from the conditionallyactive biological protein once the conditionally active biologicalprotein has reached its intended location such as a brain, synovialfluid, a tumor microenvironment, or a stem cell niche. In theseembodiments, the conditionally active biological proteins may act as adelivery vehicle for transporting the conjugated molecules (such astherapeutics or diagnostics) into a brain, synovial fluid, a tumormicroenvironment, or stem cell niches. Once inside the brain, synovialfluid, a tumor microenvironment, or stem cell niches, the conjugatedmolecule can be released for treatment of disease.

The conjugation of the conditionally active biological protein with amolecule (therapeutics or diagnostics) can be covalent conjugation ornon-covalent. Covalent conjugation can either be direct or via a linker.In certain embodiments, direct conjugation is by construction of afusion protein (i.e., by genetic fusion of the two genes encoding theconditionally active antibody and neurological disorder drug andexpression as a single protein). In certain embodiments, directconjugation is by formation of a covalent bond between a reactive groupon one of the two portions of the conditionally active antibody and acorresponding group or acceptor on the neurological drug/imaging agent.In certain embodiments, direct conjugation is by modification (i.e.,genetic modification) of one of the two molecules to be conjugated toinclude a reactive group (as non-limiting examples, a sulfhydryl groupor a carboxyl group) that forms a covalent attachment to the othermolecule to be conjugated under appropriate conditions. As onenon-limiting example, a molecule (i.e., an amino acid) with a desiredreactive group (i.e., a cysteine residue) may be introduced into, e.g.,the conditionally active antibody and a disulfide bond formed with theneurological drug. Methods for covalent conjugation of nucleic acids toproteins are also known in the art (i.e., photocrosslinking, see, e.g.,Zatsepin et al. Russ. Chem. Rev., 74: 77-95 (2005)) Non-covalentconjugation can be by any non-covalent attachment means, includinghydrophobic bonds, ionic bonds, electrostatic interactions, and thelike, as will be readily understood by one of ordinary skill in the art.

Conjugation may also be performed using a variety of linkers. Forexample, a conditionally active antibody and a neurological drug may beconjugated using a variety of bifunctional protein coupling agents suchas N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Peptide linkers, comprised of from oneto twenty amino acids joined by peptide bonds, may also be used. Incertain such embodiments, the amino acids are selected from the twentynaturally-occurring amino acids. In certain other such embodiments, oneor more of the amino acids are selected from glycine, alanine, proline,asparagine, glutamine and lysine. The linker may be a “cleavable linker”facilitating release of the neurological drug upon delivery to thebrain. For example, an acid-labile linker, peptidase-sensitive linker,photolabile linker, dimethyl linker or disulfide-containing linker(Chari et al., Cancer Res., 52:127-131 (1992); U.S. Pat. No. 5,208,020)may be used. Some examples of cross-linker reagents for antibodyconjugation include BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP,SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS,sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate).

The conjugated therapeutic agent may be toxic to the body, such as aradioactive particle, chemotherapy drug, or a cell toxin (i.e.,cytotoxin). These therapeutic agents are highly toxic to the body. Usingthe conditionally active antibodies of the present invention to deliverthe conjugated therapeutic agent to the disease site can significantlyreduce the toxic effects of these therapeutic agents. The technology forconjugating radioactive particles to antibodies is known in the art.Ibritumomab tiuxetan (Zevalin®) and tositumomab (Bexxar®) are examplesof radioactive particle conjugated monoclonal antibodies. Both areantibodies against the CD20 antigen conjugated with a differentradioactive particle. Similarly, the technology for conjugatingchemotherapy drugs to antibodies is also known in the art. There are twomarketed antibodies that are conjugated with a chemotherapy drug:brentuximab vedotin (Adcetris®) and ado-trastuzumab emtansine(Kadcyla™). Brentuximab vedotin is made up of an antibody that targetsthe CD30 antigen (found on B cells and T cells), attached to a chemodrug called MMAE. Ado-trastuzumab emtansine is made of an antibody thattargets the HER2 protein attached to a chemotherapy drug called DM1. Thetechnology for conjugating a cell toxin to an antibody is also known inthe art. For example, denileukin diftitox (Ontak®, a cancer drug)consists of an immune system protein known as interleukin-2 (IL-2)attached to a toxin from the germ that causes diphtheria.

It is contemplated that any radioactive particles, chemotherapy drugs,and cell toxins may be conjugated to the conditionally active biologicalproteins of the present invention in order to reduce the side effects ofthese agents.

In some embodiments, the radioactive particles conjugated to theconditionally active biological proteins for treatment of an abnormaltissue comprise particles impregnated with one or more radioactiveisotopes, and have sufficient radioactivity for locoregional ablation ofcells in the abnormal tissue. The particles may comprise glass, metal,resin, albumin, or polymer. Metal in the radioactive particles may beselected from iron, gadolinium, and calcium. Examples of the one or moreradioactive isotopes in the radioactive particles are selected from thegroup consisting of Gallium-67 (⁶⁷Ga), Yttrium-90 (⁹⁰Y), Gallium-68(⁶⁸Ga), Thallium-201 (²⁰¹Tl), Strontium-89 (⁸⁹Sr), Indium-III(^(III)In), Iodine-131 (¹³¹I), Samarium-153 (¹⁵³Sm), Technetium-99m(^(99m)Tc), Rhenium-186 (¹⁸⁶Re), Rhenium-188 (¹⁸⁸Re), Copper-62 (⁶²Cu),and Copper-64 (⁶⁴Cu). Preferably the radioactive isotope(s) in thecomposition emit beta radiations, gamma radiations, and/or positrons.

In some embodiments, the chemotherapy drugs conjugated to theconditionally active biological proteins are selected from the groupconsisting of anthracyclines, topoisomerase I and/or II inhibitors,spindle poison plant alkaloids, alkylating agents, anti-metabolites,ellipticine and harmine.

Anthracyclines (or anthracycline antibiotics) are derived fromStreptomyces bacteria. These compounds are used to treat a wide range ofcancers, including for example hepatocellular carcinoma, leukemias,lymphomas, and breast, uterine, ovarian, and lung cancers.Anthracyclines include, but are not limited to doxorubicin,daunorubicin, epirubicin, idarubicin, valrubicin, pirarubicin,zorubicin, aclarubicin, detorubicin, carminomycin,morpholinodoxorubicin, morpholinodaunorubicin,methoxymorpholinyldoxorubicin, and their pharmaceutically acceptablesalts thereof.

Topoisomerases are essential enzymes that maintain the topology of DNA.Inhibition of type I or type II topoisomerases interferes with bothtranscription and replication of DNA by upsetting proper DNAsupercoiling. Some type I topoisomerase inhibitors include camptothecinsderivatives Camptothecin derivatives refer to camptothecin analogs suchas irinotecan, topotecan, hexatecan, silatecan, lutortecan, karenitecin(BNP1350), gimatecan (ST1481), belotecan (CKD602), or theirpharmaceutically acceptable salts. Examples of type II topoisomeraseinhibitors include, but are not limited to, amsacrine, etoposide,etoposide phosphate and teniposide. These are semisynthetic derivativesof epipodophyllotoxins, alkaloids naturally occurring in the root ofAmerican Mayapple (Podophyllum peltatum).

Spindle poison plant alkaloids are derived from plants and block celldivision by preventing microtubule function, essential for celldivision. These alkaloids include, but are not limited to, vincaalkaloids (like vinblastine, vincristine, vindesine, vinorelbine andvinpocetine) and taxanes. Taxanes include, but are not limited to,paclitaxel, docetaxel, larotaxel, cabazitaxel, ortataxel, tesetaxel, andtheir pharmaceutically acceptable salts.

Alkylating agents are so named because of their ability to add alkylgroups to many electronegative groups under conditions present in cells.They impair cell function by forming covalent bonds with the amino,carboxyl, sulfhydryl, and phosphate groups in biologically importantmolecules. Noteworthy, their cytotoxicity is thought to result frominhibition of DNA synthesis. Alkylating agents include, but are notlimited to, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamideand platinum compounds such as oxaliplatin, cisplatin or carboplatin.

An anti-metabolite is a chemical that inhibits the use of a metabolite,which is part of normal metabolism. Such substances are often similar instructure to the metabolite that they interfere with. The presence ofanti-metabolites halts cell growth and cell division.

Purine or pyrimidine analogues prevent the incorporation of nucleotidesinto DNA, stopping DNA synthesis and thus cell divisions. They alsoaffect RNA synthesis. Examples of purine analogues include azathioprine,mercaptopurine, thioguanine, fludarabine, pentostatin and cladribine.Examples of pyrimidine analogues include 5-fluorouracil (5FU), whichinhibits thymidylate synthase, floxuridine (FUDR) and cytosinearabinoside (Cytarabine).

Antifolates are chemotherapy drugs which impair the function of folicacids. A well-known example is methotrexate, which is a folic acidanalogue that inhibits the enzyme dihydrofolate reductase (DHFR), andthus prevents the formation of tetrahydrofolate. Tetrahydrofolate isessential for purine and pyrimidine synthesis. This leads to inhibitedproduction of DNA, RNA and proteins (as tetrahydrofolate is alsoinvolved in the synthesis of amino acids serine and methionine). Otherantifolates include, but are not limited to, trimethoprim, raltitrexed,pyrimethamine and pemetrexed.

Other chemotherapy drugs may also be conjugated to the conditionallyactive biological proteins, such as ellipticine and harmine. Ellipticineis a natural plant alkaloid product which is isolated from the evergreentree of the Apocynaceae family. Ellipticine and its derivatives such as9-hydroxyellipticinium, N2-methyl-9-hydroxyellipticinium,2-(diethyiamino-2-ethyl)9-hydroxyellipticinium acetate,2-(diisopropylamino-ethyl)9-hydroxy-ellipticinium acetate and 2-(betapiperidino-2-ethyl)9-hydroxyellipticinium are all effective chemotherapydrugs.

Harmine is a natural plant alkaloid product which was isolated from thePeganum harmala seeds. Harmine-based chemotherapy drugs include harmine,harmaline, harmol, harmalol and harman, and quinazoline derivatives:vasicine and vasicinone.

In some embodiments, the cell toxins conjugated to the conditionallyactive biological proteins include taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracinedione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Other toxinsinclude, for example, ricin, CC-1065 and analogues, the duocarmycins.Still other toxins include diptheria toxin, and snake venom (e.g., cobravenom).

In one embodiment, a pyrrolobenzodiazepine may be conjugated to aconditionally active biological protein. Pyrrolobenzodiazepine (PBD)dimers are a class of rationally designed DNA minor groove, sequenceselective, cross-linking agents, which cross-link the two DNA strandsthus preventing DNA replication and cell division. The PBDs may be usedas chemotherapy agents. This class of chemotherapy agents exhibitspicomolar or subpicomolar activity in inhibiting tumor cell growth. Thesynthetic PBDs, when conjugated to a conditionally active antibody, canbe guided towards a tumor site for inhibition of tumor cell growth. PBDsmay use different conjugation sites for linking to a conditionallyactive antibody. For example, two suitable PBDs are show below.

In some embodiments, the conditionally active biological proteins of thepresent invention may be conjugated to a diagnostic agent. A diagnosticagent used in the present invention can include any diagnostic agentknown in the art, as provided, for example, in the following references:Armstrong et al, Diagnostic Imaging, 5^(th) Ed., Blackwell Publishing(2004); Torchilin, V. P., Ed., Targeted Delivery of Imaging Agents, CRCPress (1995); Vallabhajosula, S., Molecular Imaging:Radiopharmaceuticals for PET and SPECT, Springer (2009). A diagnosticagent can be detected by a variety of methods, including using the agentto provide and/or enhance a detectable signal that includes, but is notlimited to, gamma-emitting, radioactive, echogenic, optical,fluorescent, absorptive, magnetic or tomography signals. Techniques forimaging the diagnostic agent can include, but are not limited to, singlephoton emission computed tomography (SPECT), magnetic resonance imaging(MRI), optical imaging, fluorescence imaging, positron emissiontomography (PET), computed tomography (CT), x-ray imaging, gamma rayimaging, and the like.

In some embodiments, a diagnostic agent can include chelators that bind,e.g., to metal ions to be used for a variety of diagnostic imagingtechniques. Exemplary chelators include but are not limited toethylenediaminetetraacetic acid (EDTA),[4-(1,4,8,11-tetraazacyclotetradec-1-yl) methyljbenzoic acid (CPTA),Cyclohexanediaminetetraacetic acid (CDTA),ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA),diethylenetriaminepentaacetic acid (DTPA), citric acid, hydroxyethylethylenediamine triacetic acid (HEDTA), iminodiacetic acid (IDA),triethylene tetraamine hexaacetic acid (TTHA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic acid)(DOTP), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), andderivatives thereof.

A radioisotope can be incorporated into some of the diagnostic agentsdescribed herein and can include radionuclides that emit gamma rays,positrons, beta and alpha particles, and X-rays. Suitable radionuclidesinclude but are not limited to Ac, As, At, ^(n)B, ¹²⁸Ba, ²¹²Bi, ⁷⁵Br,⁷⁷Br, ¹⁴C, ¹⁰⁹Cd, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁸F, ⁶⁷Ga, ⁶⁸Ga, ³H, ¹²³I, ¹²⁵I,¹³⁰I, ¹³¹I, ¹¹¹In, ¹⁷⁷Lu, ¹³N, ¹⁵0, ³²P, ³³P, ²¹²Pb, ¹⁰³Pd, ¹⁸⁶Re,¹⁸⁸Re, ⁴⁷Sc, ¹⁵³Sm, ⁸⁹Sr, ¹¹¹Tc, ⁸⁸Y and ⁹⁰Y. In certain embodiments,radioactive agents can include ^(m)In-DTPA, ^(99m)Tc(CO)3-DTPA,^(99m)Tc(CO)₃-ENPy2, ^(62/64/67)Cu-TETA, ^(99m)Tc(CO)₃-IDA, and^(99m)Tc(CO)₃triamines (cyclic or linear). In other embodiments, theagents can include DOTA and its various analogs with ¹¹¹In, ¹⁷⁷Lu,¹⁵³Sm, ^(88/90)Y, ^(62/64/67)Cu, or ^(67/68)Ga. In some embodiments, theliposomes can be radiolabeled, for example, by incorporation of lipidsattached to chelates, such as DTPA-lipid, as provided in the followingreferences: Phillips et al, Wiley Interdisciplinary Reviews:Nanomedicine and Nanobiotechnology, vol. 1, pages 69-83 (2008);Torchilin, V. P. & Weissig, V., Eds. Liposomes 2nd Ed. : Oxford Univ.Press (2003); Elbayoumi, T. A. & Torchilin, V. P., Eur. J. Nucl. Med.Mol. Imaging, 33: 1196-1205 (2006); Mougin-Degraef, M. et al, Int'l J.Pharmaceutics, 344: 110-1 17 (2007).

In other embodiments, the diagnostic agents may include optical agentssuch as fluorescent agents, phosphorescent agents, chemiluminescentagents, and the like. Numerous agents (e.g., dyes, probes, labels, orindicators) are known in the art and can be used in the presentinvention. (See, e.g., Invitrogen, The Handbook—A Guide to FluorescentProbes and Labeling Technologies, Tenth Edition (2005)). Fluorescentagents can include a variety of organic and/or inorganic small moleculesor a variety of fluorescent proteins and derivatives thereof. Forexample, fluorescent agents can include but are not limited to cyanines,phthalocyanines, porphyrins, indocyanines, rhodamines, phenoxazines,phenylxanthenes, phenothiazines, phenoselenazines, fluoresceins,benzoporphyrins, squaraines, dipyrrolo pyrimidones, tetracenes,quinolines, pyrazines, corrins, croconiums, acridones, phenanthridines,rhodamines, acridines, anthraquinones, chalcogenopyrylium analogues,chlorins, naphthalocyanines, methine dyes, indolenium dyes, azocompounds, azulenes, azaazulenes, triphenyl methane dyes, indoles,benzoindoles, indocarbocyanines, benzoindocarbocyanines, and BODIPY™derivatives having the general structure of4,4-difiuoro-4-bora-3a,4a-diaza-s-indacene, and/or conjugates and/orderivatives of any of these. Other agents that can be used include, butare not limited to, fluorescein, fluorescein-polyaspartic acidconjugates, fluorescein-polyglutamic acid conjugates,fluorescein-polyarginine conjugates, indocyanine green,indocyanine-dodecaaspartic acid conjugates, indocyanine(NIRD)-polyaspartic acid conjugates, isosulfan blue, indoledisulfonates, benzoindole disulfonate,bis(ethylcarboxymethyl)indocyanine, bis(pentylcarboxymethyl)indocyanine,polyhydroxyindole sulfonates, polyhydroxybenzoindole sulfonate, rigidheteroatomic indole sulfonate, indocyaninebispropanoic acid,indocyaninebishexanoic acid,3,6-dicyano-2,5-[(N,N,N′,N′-tetrakis(carboxymethyl)amino]pyrazine,3,6-[(N,N,N′,N′-tetrakis(2-hydroxyethyl)amino]pyrazine-2,5-dicarboxylicacid, 3,6-bis(N-azatedino)pyrazine-2,5-dicarboxylic acid,3,6-bis(N-morpholino)pyrazine-2,5-dicarboxylic acid,3,6-bis(N-piperazino)pyrazine-2,5-dicarboxylic acid,3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid,3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid S-oxide,2,5-dicyano-3,6-bis(N-thiomorpholino)pyrazine S,S-dioxide,indocarbocyaninetetrasulfonate, chloroindocarbocyanine, and3,6-diaminopyrazine-2,5-dicarboxylic acid.

In yet other embodiments, the diagnostic agents may include contrastagents that are generally well known in the art, including, for example,superparamagnetic iron oxide (SPIO), complexes of gadolinium ormanganese, and the like. (See, e.g., Armstrong et al, DiagnosticImaging, 5^(th) Ed., Blackwell Publishing (2004)). In some embodiments,a diagnostic agent can include a magnetic resonance (MR) imaging agent.Exemplary magnetic resonance agents include but are not limited toparamagnetic agents, superparamagnetic agents, and the like. Exemplaryparamagnetic agents can include but are not limited to Gadopenteticacid, Gadoteric acid, Gadodiamide, Gadolinium, Gadoteridol ,Mangafodipir, Gadoversetamide, Ferric ammonium citrate, Gadobenic acid,Gadobutrol, or Gadoxetic acid. Superparamagnetic agents can include butare not limited to superparamagnetic iron oxide and Ferristene. Incertain embodiments, the diagnostic agents can include x-ray contrastagents as provided, for example, in the following references: H. SThomsen, R. N. Muller and R. F. Mattrey, Eds., Trends in Contrast Media,(Berlin: Springer-Verlag, 1999); P. Dawson, D. Cosgrove and R. Grainger,Eds., Textbook of Contrast Media (ISIS Medical Media 1999); Torchilin,V. P., Curr. Pharm. Biotech., vol. 1, pages 183-215 (2000); Bogdanov, A.A. et al, Adv. Drug Del. Rev., Vol. 37, pages 279-293 (1999); Sachse, A.et al., Investigative Radiology, vol. 32, pages 44-50 (1997). Examplesof x-ray contrast agents include, without limitation, iopamidol,iomeprol, iohexol, iopentol, iopromide, iosimide, ioversol, iotrolan,iotasul, iodixanol, iodecimol, ioglucamide, ioglunide, iogulamide,iosarcol, ioxilan, iopamiron, metrizamide, iobitridol and iosimenol. Incertain embodiments, the x-ray contrast agents can include iopamidol,iomeprol, iopromide, iohexol, iopentol, ioversol, iobitridol, iodixanol,iotrolan and iosimenol.

In some embodiments, the conditionally active biological proteins may beconjugated to another protein, such as interleukins, cytokines, enzymes,growth factors, or other antibodies. Some examples of such proteinsinclude, for example, tumor necrosis factor, α-interferon (EFN-α),β-interferon (IFN-β), nerve growth factor (NGF), platelet derived growthfactor (PDGF), tissue plasminogen activator (TPA), an apoptotic agent(e.g., TNF-α, TNF-β, AIM I as disclosed in WO 97/33899), AIM II (see WO97/34911), Fas Ligand (Takahashi et al., J. Immunol., vol. 6, pages1567-1574, 1994), and VEGI (WO 99/23105), a thrombotic agent or ananti-angiogenic agent (e.g., angiostatin or endostatin); or a biologicalresponse modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-I”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), or a growth factor(e.g., growth hormone (“GH”)).

In some embodiments, the conditionally active antibodies for crossingthe BBB as provided by the present invention may be conjugated to a drugfor treating a neurological disorder. The drug will be transportedacross the BBB with the antibodies and remain in the brain for treatingthe neurological disorder. The neurological disorder refers to a diseaseor disorder which affects the CNS and/or which has an etiology in theCNS. Exemplary CNS diseases or disorders include, but are not limitedto, neuropathy, amyloidosis, cancer, an ocular disease or disorder,viral or microbial infection, inflammation, ischemia, neurodegenerativedisease, seizure, behavioral disorders, and a lysosomal storage disease.For the purposes of this application, the CNS will be understood toinclude the eye, which is normally sequestered from the rest of the bodyby the blood-retina barrier. Specific examples of neurological disordersinclude, but are not limited to, neurodegenerative diseases (including,but not limited to, Lewy body disease, postpoliomyelitis syndrome,Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease,multiple system atrophy, striatonigral degeneration, tauopathies(including, but not limited to, Alzheimer disease and supranuclearpalsy), prion diseases (including, but not limited to, bovine spongiformencephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru,Gerstmann-Straussler-Scheinker disease, chronic wasting disease, andfatal familial insomnia), bulbar palsy, motor neuron disease, andnervous system heterodegenerative disorders (including, but not limitedto, Canavan disease, Huntington's disease, neuronalceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkeskinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome,lafora disease, Rett syndrome, hepatolenticular degeneration,Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia(including, but not limited to, Pick's disease, and spinocerebellarataxia), cancer (e.g. of the CNS and/or brain, including brainmetastases resulting from cancer elsewhere in the body).

The drugs for treating the neurological disorder include, but are notlimited to, antibodies, peptides, proteins, natural ligands of one ormore CNS target(s), modified versions of natural ligands of one or moreCNS target(s), aptamers, inhibitory nucleic acids (i.e., smallinhibitory RNAs (siRNA) and short hairpin RNAs (shRNA)), ribozymes, andsmall molecules, or active fragments of any of the foregoing. Exemplaryneurological disorder drugs include, but are not limited to: antibodies,aptamers, proteins, peptides, inhibitory nucleic acids and smallmolecules and active fragments of any of the foregoing that either arethemselves or specifically recognize and/or act upon (i.e., inhibit,activate, or detect) a CNS antigen or target molecule such as, but notlimited to, amyloid precursor protein or portions thereof, amyloid beta,beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin,huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers,and neurotrophins. Non-limiting examples of neurological disorder drugsand disorders they may be used to treat include anti-BACE1 antibody fortreating Alzheimer's, acute and chronic brain injury, stroke; anti-Abetaantibody for treating Alzheimer's disease; neurotrophin for treatingstroke, acute brain injury, spinal cord injury; brain-derivedneurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF-2) fortreating chronic brain injury (neurogenesis); anti-Epidermal GrowthFactor Receptor (EGFR)-antibodies for treating brain cancer; Glialcell-line derived neural factor (GDNF) for treating Parkinson's disease;brain-derived neurotrophic factor (BDNF) for treating Amyotrophiclateral sclerosis and depression; lysosomal enzyme for treatinglysosomal storage disorders of the brain; Ciliary neurotrophic factor(CNTF) for treating Amyotrophic lateral sclerosis; Neuregulin-1 fortreating Schizophrenia; and anti-HER2 antibody (e.g. trastuzumab) fortreating brain metastasis from HER2-positive cancer.

In some embodiments, the conjugation of the conditionally activebiological proteins may be on the Fc region of the antibodies. Theconjugating molecules, compound or drugs described above may beconjugated to the Fc region, as described in U.S. Pat. No. 8,362,210(incorporated herein by reference). For example, Fc region may beconjugated to a cytokine or a toxin to be delivered to the site wherethe conditionally active antibody displays preferentially activity.Methods for conjugating polypeptides to the Fc region of antibodies areknown in the art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929,5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626,5,844,095, and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCTpublications WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO99/04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA, vol. 88, pages10535-10539, 1991; Traunecker et al., Nature, vol. 331, pages 84-86,1988; Zheng et al., J. Immunol., vol. 154, pages 5590-5600, 1995; andViI et al., Proc. Natl. Acad. Sci. USA, vol. 89, pages 11337-11341,1992, which are incorporated herein by reference in their entireties.

Engineering Multispecific Conditionally Active Antibodies

When the conditionally active biological proteins are conditionallyactive antibodies, the conditionally active antibodies may be engineeredto generated multispecific conditionally active antibodies. Themultispecific antibody is an antibody with polyepitopic specificity, asdescribed in WO 2013/170168, incorporated herein by reference in itsentirety. Multispecific antibodies include, but are not limited to, anantibody comprising a heavy chain variable domain (V_(H)) and a lightchain variable domain (VL), where the V_(H)V_(L) unit has polyepitopicspecificity, antibodies having two or more V_(L) and V_(H) domains whereeach V_(H)V_(L) unit binds to a different epitope, antibodies having twoor more single variable domains with each single variable domain bindingto a different epitope, and antibodies comprising one or more antibodyfragments as well as antibodies comprising antibody fragments that havebeen linked covalently or non-covalently.

To construct multispecific antibodies, including bispecific antibodies,antibody fragments having at least one free sulfhydryl group areobtained. The antibody fragments may be obtained from full-lengthconditionally active antibodies. The conditionally active antibodies maybe digested enzymatically to produce antibody fragments. Exemplaryenzymatic digestion methods include, but are not limited to, pepsin,papain and Lys-C. Exemplary antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)2, Fv, diabodies (Db); tandem diabodies(taDb), linear antibodies (see U.S. Pat. No. 5,641,870, Example 2;Zapata et al., Protein Eng., vol. 8, pages 1057-1062 (1995)); one-armedantibodies, single variable domain antibodies, minibodies (Olafsen et al(2004) Protein Eng. Design & Sel., vol. 17, pages 315-323), single-chainantibody molecules, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, CDR (complementary determiningregion), and epitope-binding fragments. Antibody fragments may also beproduced using DNA recombinant technology. The DNA encoding the antibodyfragments may be cloned into plasmid expression vectors or phagemidvectors and expressed directly in E. Coli. Antibody enzymatic digestionmethods, DNA cloning and recombinant protein expression methods are wellknown to those skilled in the art.

Antibody fragments may be purified using conventional techniques and aresubjected to reduction to generate a free thiol group. Antibodyfragments having a free thiol group are reacted with a crosslinker, forexample, bis-maleimide. Such crosslinked antibody fragments are purifiedand then reacted with a second antibody fragment having a free thiolgroup. The final product in which two antibody fragments are crosslinkedis purified. In certain embodiments, each antibody fragment is a Fab andthe final product, in which the two Fabs are linked throughbis-maleimide, is referred to herein as bismaleimido-(thio-Fab)2, orbis-Fab. Such multispecific antibodies and antibody analogs, includingbis-Fabs, can be exploited to quickly synthesize a large number ofantibody fragment combinations, or structural variants of nativeantibodies or particular antibody fragment combinations.

Multispecific antibodies can be synthesized with modified crosslinkerssuch that additional functional moieties may be attached to themultispecific antibody. Modified crosslinkers allow for attachment ofany sulfhydryl-reactive moiety. In one embodiment,N-succinimidyl-S-acetylthioacetate (SAT A) is attached to bis-maleimideto form bis-maleimido-acetylthioacetate (BMata). After deprotection ofthe masked thiol group, any functional group having asulfhydryl-reactive (or thiol-reactive) moiety may be attached to themultispecific antibodies.

Exemplary thiol-reactive reagents include a multifunctional linkerreagent, a capture, i.e. affinity, label reagent (e.g. a biotin-linkerreagent), a detection label (e.g. a fluorophore reagent), a solid phaseimmobilization reagent (e.g. SEPHAROSE™, polystyrene, or glass), or adrug-linker intermediate. One example of a thiol-reactive reagent isN-ethyl maleimide (NEM). Such multispecific antibodies or antibodyanalogs having modified crosslinkers may be further reacted with a drugmoiety reagent or other label. Reaction of a multispecific antibody orantibody analog with a drug-linker intermediate provides a multispecificantibody drug conjugate or antibody analog drug conjugate, respectively.

Many other techniques for making multispecific antibodies may also beused in the present invention. References (incorporated herein byreferences) describing these techniques include: (1) Milstein andCuello, Nature, vol. 305, page 537 (1983)), WO 93/08829, and Trauneckeret al., EMBO J., vol. 10, page 3655 (1991) on recombinant co-expressionof two immunoglobulin heavy chain-light chain pairs having differentspecificities; (2) U.S. Pat. No. 5,731,168 on “knob-in-hole”engineering; (3) WO 2009/089004A1 on engineering electrostatic steeringeffects for making antibody Fc-heterodimeric molecules; (4) U.S. Pat.No. 4,676,980, and Brennan et al., Science, vol. 229, page 81 (1985) oncross-linking two or more antibodies or fragments; (5) Kostelny et al.,J. Immunol., vol. 148, pages 1547-1553 (1992) on using leucine zippersto produce bi-specific antibodies; (6) Hollinger et al., Proc. Natl.Acad. Sci. USA, vol. 90, pages 6444-6448 (1993) on using “diabody”technology for making bispecific antibody fragments; (7) Gruber et al.,J. Immunol., vol. 152, page 5368 (1994) on using single-chain Fv (sFv)dimers; (8) Tutt et al. J. Immunol. 147: 60 (1991) on preparingtrispecific antibodies; and (9) US 2006/0025576A1 and Wu et al. NatureBiotechnology, vol. 25, pages 1290-1297 (2007) on engineered antibodieswith three or more functional antigen binding sites, including “Octopusantibodies” or “dual-variable domain immunoglobulins” (DVDs).

Multispecific antibodies of the present invention might also begenerated as described in WO/2011/109726, incorporated herein byreference in its entirety.

In one embodiment, the conditionally active antibody for crossing theBBB is engineered to make a multispecific antibody (e.g. a bispecificantibody). This multispecific antibody comprises a first antigen bindingsite which binds a BBB-R and a second antigen binding site which binds abrain antigen. At least the first antigen binding site for BBB-R isconditionally active. A brain antigen is an antigen expressed in thebrain, which can be targeted with an antibody or small molecule.Examples of such antigens include, without limitation: beta-secretase 1(BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR),human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4(ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucinerich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gammasecretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75neurotrophin receptor (p75NTR), and caspase 6. In one embodiment, theantigen is BACE1.

Multispecific antibodies have high selectivity at preferentiallytargeting tissues containing all or most of the targets (antigens) thata multispecific antibody can bind to. For example, a bispecific antibodyprovides selectivity for target cells by displaying greater preferenceto target cells that express both of the antigens recognized by thebispecific antibody, in comparison with non-target cells that mayexpress only one of the antigens. Therefore, due to the dynamism of thesystem, there are more bispecific antibodies being bound to the targetcells than non-target cells at equilibrium.

Engineering the Fc Region of Conditionally Active Antibodies

When the conditionally active biological proteins are conditionallyactive antibodies, the conditionally active antibodies may be engineeredat their fragment crystallizable region (Fc region). The Fc region isthe tail region of an antibody that interacts with cell surfacereceptors called Fc receptors and some proteins of the complementsystem. Unlike the Fab region that is specific for each antigen, the Fcregion of all antibodies in a class is the same for each speciesregardless which antigen the antibody binds.

The Fc receptors are members of the immunoglobulin gene superfamily ofproteins. Fc receptors are found on a number of cells in the immunesystem including phagocytes like macrophages and monocytes, granulocyteslike neutrophils and eosinophils, and lymphocytes of the innate immunesystem (natural killer cells) or adaptive immune system (e.g., B cells).After binding with an antibody, the Fc receptor activates these cellsand allows these cells to identify and eliminate antigens (such asmicrobial pathogens) that are bound on the Fab region of the antibody.The Fc receptor mediated killing mechanisms include complement-dependentcytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), andantibody-dependent cellular phagocytosis (ADCP).

In some embodiments, the Fc region is engineered to introduce mutationssuch as amino acid substitutions in the Fc region. Such substitution inthe Fc region may increase the half-life of the mutated antibody inserum. For example, the half-life of an IgG antibody is correlated withits pH-dependent binding to neonatal receptor FcRn, which is expressedon the surface of endothelial cells and protects the IgG in apH-dependent manner from degradation. Several amino acid substitutionsat the Fc region, such as T250Q/M428L and M252Y/S254T/T256E+H433K/N434F,have shown increased binding affinity of the antibody to FcRn and extendthe half-life of the antibody.

Amino acid substitutions may also be introduced to the Fc region toalter effector functions. For example, human antibodies in the IgG classbind to Fcy receptors (FcγRI, FcγRIIa, FcγRIIIa), the inhibitory FcγRIIbreceptor, and the first component of complement (C1q) with differentaffinities, yielding very different effector functions among differentantibodies. Binding of IgG antibody to FcγRs or C1q depends on residueslocated in the hinge domain and the CH2 domain of the antibody. Aminoacid substitutions in human antibodies IgG1 or IgG2 residues atpositions 233-236 and antibody IgG4 residues at positions 327, 330 and331 can greatly reduce ADCC and CDC. Furthermore, alanine substitutionat different positions in the Fc region, including K322, significantlyreduced complement activation. Many more examples of engineering the Fcregion are described in U.S. Pat. No. 8,362,210, which is incorporatedby reference in its entirety.

In some embodiments, the Fc region of an antibody may be engineered tobe capable of recognizing an antigen (US 2010/0256340, incorporatedherein by reference). At least one, preferably two, extra Fab fragmentsmay be linked onto the Fc region of an antibody. In some embodiments,the extra Fab fragments are conditionally active. For example, theantibody of the present invention for crossing the BBB may contain suchan extra Fab fragment with affinity for a BBB-R on the plasma side andlittle or no affinity to the BBB-R on the brain side. The antibody canalso bind to multiple brain antigens, thus may have a higher selectivityfor preferentially acting on sites where these antigens are present.

Pharmaceutical Compositions

The present disclosure provides at least one composition comprising (a)a conditionally active biologic protein; and (b) a suitable carrier ordiluent. The present disclosure also provides at least one compositioncomprising (a) a conditionally active biologic protein encoding nucleicacid as described herein; and (b) a suitable carrier or diluent. Thecarrier or diluent can optionally be pharmaceutically acceptable,according to known carriers or diluents. The composition can optionallyfurther comprise at least one further compound, protein or composition.In some embodiment, the conditionally active biologic protein is aconditionally active antibody.

The conditionally active biologic protein may be in the form of apharmaceutically acceptable salt. Pharmaceutically acceptable saltsmeans which can be generally used as salts of an therapeutic protein inpharmaceutical industry, including for example, salts of sodium,potassium, calcium and the like, and amine salts of procaine,dibenzylamine, ethylenediamine, ethanolamine, methylglucamine, taurine,and the like, as well as acid addition salts such as hydrochlorides, andbasic amino acids and the like.

The present disclosure further provides at least one conditionallyactive biologic protein method or composition, for administering atherapeutically effective amount to modulate or treat at least oneparent molecule related condition in a cell, tissue, organ, animal orpatient and/or, prior to, subsequent to, or during a related condition,as known in the art and/or as described herein. Thus, the disclosureprovides a method for diagnosing or treating a condition associated withthe wild-type protein in a cell, tissue, organ or animal, comprisingcontacting or administering a composition comprising an effective amountof at least one conditionally active biologic protein of the disclosurewith, or to, the cell, tissue, organ or animal. The method canoptionally further comprise using an effective amount of 0.001-50mg/kilogram of a conditionally active biologic protein of the disclosureto the cells, tissue, organ or animal. The method can optionally furthercomprise using the contacting or the administrating by at least one modeselected from parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal. Themethod can optionally further comprise administering, prior,concurrently, or after the conditionally active biologic proteincontacting or administering at least one composition comprising aneffective amount of at least one compound or protein selected from atleast one of a detectable label or reporter, a TNF antagonist, anantirheumatic, a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAK)), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial, anantipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin,an immunization, an immunoglobulin, an immunosuppressive, a growthhormone, a hormone replacement drug, a radiopharmaceutical, anantidepressant, an antipsychotic, a stimulant, an asthma medication, abeta agonist, an inhaled steroid, an epinephrine or analog thereof, acytotoxic or other anti-cancer agent, an anti-metabolite such asmethotrexate, or an antiproliferative agent.

The present disclosure further provides at least one conditionallyactive biologic protein method for diagnosing at least one wild-typeprotein related condition in a cell, tissue, organ, animal or patientand/or, prior to, subsequent to, or during a related condition, as knownin the art and/or as described herein.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of conditionally active biologicprotein in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe patient's needs.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Pharmaceuticalcompositions and formulations of the invention for oral administrationcan be formulated using pharmaceutically acceptable carriers well knownin the art in appropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropyhnethyl cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Tablet forms can include one or moreof lactose, sucrose, mannitol, sorbitol, calcium phosphates, cornstarch, potato starch, tragacanth, microcrystalline cellulose, acacia,gelatin, colloidal silicon dioxide, croscannellose sodium, talc,magnesium stearate, stearic acid, and other excipients, colorants,fillers, binders, diluents, buffering agents, moistening agents,preservatives, flavoring agents, dyes, disintegrating agents, andpharmaceutically acceptable carriers.

The invention provides aqueous suspensions comprising a conditionallyactive biologic protein, in admixture with excipients suitable for themanufacture of aqueous suspensions. Such excipients include a suspendingagent, such as sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolality.

Lozenge forms can comprise the active ingredient in a flavor, usuallysucrose and acacia or tragacanth, as well as pastilles comprising theactive ingredient in an inert base, such as gelatin and glycerin orsucrose and acacia emulsions, gels, and the like containing, in additionto the active ingredient, carriers known in the art. It is recognizedthat the conditionally active biologic protein, when administeredorally, must be protected from digestion. This is typically accomplishedeither by complexing the conditionally active biologic protein with acomposition to render it resistant to acidic and enzymatic hydrolysis orby packaging the conditionally active biologic protein in anappropriately resistant carrier such as a liposome. Means of protectingproteins from digestion are well known in the art. The pharmaceuticalcompositions can be encapsulated, e.g., in liposomes, or in aformulation that provides for slow release of the active ingredient.

The packaged conditionally active biologic protein, alone or incombination with other suitable components, can be made into aerosolformulations (e.g., they can be “nebulized”) to be administered viainhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. Suitable formulations for rectal administrationinclude, for example, suppositories, which consist of the packagednucleic acid with a suppository base. Suitable suppository bases includenatural or synthetic triglycerides or paraffin hydrocarbons, inaddition, it is also possible to use gelatin rectal capsules whichconsist of a combination of the packaged nucleic acid with a base,including, for example, liquid triglycerides, polyethylene glycols, andparaffin hydrocarbons.

Dermal or topical delivery compositions of the invention may include inaddition to a conditionally active biologic protein, a pharmaceuticallyacceptable carrier in a cream, ointment, solution or hydrogelformulation, and other compounds so long as the added component does notdeleteriously affect delivery of the therapeutic protein. Conventionalpharmaceutically acceptable emulsifiers, surfactants, suspending agents,antioxidants, osmotic enhancers, extenders, diluents and preservativesmay also be added. Water soluble polymers can also be used as carriers.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives,in the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. In one aspect, parenteral modes ofadministration are preferred methods of administration for compositionscomprising a conditionally active biologic protein. The compositions mayconveniently be administered in unit dosage form and may be prepared byany of the methods well-known in the pharmaceutical art, for example asdescribed in Remington's Pharmaceutical Sciences, Mack Publishing Co.Easton Pa., 18^(th) Ed., 1990. Formulations for intravenousadministration may contain a pharmaceutically acceptable carrier such assterile water or saline, polyalkylene glycols such as polyethyleneglycol, oils of vegetable origin, hydrogenated naphthalenes and thelike. Also see and adapt the description in U.S. Pat. No. 4,318,905.

The formulations of packaged compositions comprising a conditionallyactive biologic protein can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials. Injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

The present disclosure also provides at least one conditionally activebiologic protein composition, device and/or method of delivery fordiagnosing of at least one wild-type protein related condition,according to the present disclosure.

Also provided is a composition comprising at least one conditionallyactive biologic protein and at least one pharmaceutically acceptablecarrier or diluent. The composition can optionally further comprise aneffective amount of at least one compound or protein selected from atleast one of a detectable label or reporter, a cytotoxic or otheranti-cancer agent, an anti-metabolite such as methotrexate, anantiproliferative agent, a cytokine, or a cytokine antagonist, a TNFantagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog.

Also provided is a medical device, comprising at least one conditionallyactive biologic protein of the disclosure, wherein the device issuitable to contacting or administering the at least one conditionallyactive biologic protein by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.

In a further aspect, the disclosure provides a kit comprising at leastone conditionally active biologic protein or fragment of the disclosurein lyophilized form in a first container, and an optional secondcontainer comprising sterile water, sterile buffered water, or at leastone preservative selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuricnitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesiumchloride, alkylparaben, benzalkonium chloride, benzethonium chloride,sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueousdiluent. In one aspect, in the kit, the concentration of conditionallyactive biologic protein or specified portion or variant in the firstcontainer is reconstituted to a concentration of about 0.1 mg/ml toabout 500 mg/ml with the contents of the second container, in anotheraspect, the second container further comprises an isotonicity agent. Inanother aspect, the second container further comprises a physiologicallyacceptable buffer. In one aspect, the disclosure provides a method oftreating at least one wild-type protein mediated condition, comprisingadministering to a patient in need thereof a formulation provided in akit and reconstituted prior to administration.

Also provided is an article of manufacture for human pharmaceutical ordiagnostic use, comprising packaging material and a container comprisinga solution or a lyophilized form of at least one conditionally activebiologic protein of the present disclosure. The article of manufacturecan optionally comprise having the container as a component of aparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal delivery device or system.

The present disclosure further provides any disclosure described herein.

EXAMPLE 1 General Description of a Multiwall Assay (for Example, 96-WellAssay) for Temperature Mutants

Fluorescent substrate is added to each well of a multiwall plate, atboth wild-type and new, lower reaction temperatures (for example, either37° C. or 25° C. as mentioned above) for an appropriate time period.Fluorescence is detected by measuring fluorescence in a fluorescentplate reader at appropriate excitation and emission spectra (forexample, 320 nm exitation/405 nm emission). Relative fluorescence units(RFU) are determined. Supernatant from wild type molecule andplasmid/vector transformed cells are used as positive and negativecontrols. Duplicate reactions are performed for each sample, reactiontemperature, and positive and negative control.

Mutants that are active at the lower temperature (for example, themutants active at 25° C.) and that have a decrease in activity at thewild type temperature (for example, a 10%, 20%, 30%, 40% or moredecrease in activity at 37° C.), thus having a ratio of activitiesgreater than or equal to about 1.1 or more (e.g., the ratio of theactivities at 25° C. or 37° C. (25° C./37° C.) is greater than or equalto 1.1 or more), can be deemed to be putative primary temperaturesensitive hits. These putative primary temperature sensitive hits canthen be rescreened, using the same assay, to confirm any primary hits.

EXAMPLE 2 General Description of a Different Assay Format forConfirmation of Activity (for Example, a 14-mL Assay) for TemperatureMutants

Mutants that are identified as temperature sensitive primary hits areexpressed in 14 ml culture tubes and their enzymatic activity ismeasured at wild type (for example, 37° C.) and the lower temperature(for example, 25° C.). Protein is expressed and purified as describedabove for the multiwall format, with the exception that the expressionis performed in different format (14 ml tubes) rather than the multiwall(96-well plate) format.

Each mutant supernatant is transferred to a multiwall plate, for examplea 96-well microplate. Fluorescent substrate is added to each tube at theindicated reaction temperatures (wild-type, lower temperature) for arequired period of time. Wild-type molecules are used as a positivecontrol and supernatant from cells transformed with only vector is usedas a negative control. Fluorescence is detected by measuringfluorescence in a fluorescent plate reader at the appropriate emissionspectra (for example, 320 nm exitation/405 ran emission). Relativefluorescence units (RFU) are determined. Duplicate reactions can areperformed for each sample, reaction temperature, and positive andnegative control.

Mutants that are active at the lower temperatures (for example, 25° C.)but that demonstrate at least a 30% or more decreased activity at wildtype (for example, 37° C.), thus have a ratio of activity at lowertemperature (for example, 25° C.) to wild type temperature (for example,37° C.) equal to or greater than 1.5, are identified as temperaturesensitive hits.

The activities of mutants at the lower temperature (for example 25° C.)are compared to the activity of the wild-type molecule at the wild-typetemperature (for example 37° C.). If molecules are more active than thewild-type molecules at the lower temperature (for example 25° C.), asindicated by a residual activity>1, preferably 2 or greater than 2, andif the mutants demonstrate an overall decrease in activity when comparedto the wild-type molecule at the wild-type temperature (37° C.), thephenotype of the mutants as temperature sensitive mutants can beconfirmed.

EXAMPLE 3 General Description of Further Evolution of Hits Discovered

If desired, a new, combinatorial variant library is generated from allor selected mutant hits previously identified. The new library can bedesigned to contain every possible combination of amino acid variantsfor each of the selected mutants, and rescreened as described for newhits.

It is to be understood, however, that even though numerouscharacteristics and advantages of the present invention have been setforth in the foregoing description, together with details of thestructure and function of the invention, the disclosure is illustrativeonly, and changes may be made in detail, especially in matters of shape,size and arrangement of parts within the principles of the invention tothe full extent indicated by the broad general meanings of the terms inwhich the appended claims are expressed.

What is claimed is:
 1. A method of preparing a conditionally activeantibody for crossing the blood-brain barrier, the method comprising thesteps of: i. evolving DNA which encodes a parent antibody against ablood-brain barrier receptor using one or more evolutionary techniquesto create mutant DNAs; ii. expressing the mutant DNAs to obtain mutantantibodies; iii. subjecting the mutant antibodies to an assay under afirst physiological condition in blood plasma and to an assay under asecond physiological condition in brain extracellular fluid; and iv.selecting the conditionally active antibody from the mutant antibodieswhich exhibit both (a) an affinity to the blood-brain barrier receptorin the assay under the first physiological condition, and (b) anaffinity selected from the group consisting of a decreased affinity tothe blood-brain barrier receptor in the assay under the secondphysiological condition in comparison with the affinity in the assayunder the first physiological condition and no affinity to theblood-brain barrier receptor in the assay under the second physiologicalcondition.
 2. The method of claim 1, further comprising the step ofconjugating the conditionally active antibody to a molecule.
 3. Themethod of claim 2, wherein the conjugating step comprises forming acovalent bond between the conditionally active antibody and themolecule.
 4. The method of claim 2, wherein the conjugating stepcomprises forming a non-covalent bond between the conditionally activeantibody and the molecule.
 5. The method of claim 2, wherein themolecule is selected from the group consisting of cytokines,interleukins, enzymes, hormones, growth factors, cytotoxic agents,chemotherapy drugs, radioactive particles, antibodies and diagnosticagents.
 6. The method of claim 2, wherein the molecule is conjugated tothe Fc region of the conditionally active antibody.
 7. The method ofclaim 1, further comprising the step of introducing at least one aminoacid substitution in the Fc region of the conditionally active antibody.8. The method of claim 7, wherein the at least one amino acidsubstitution is two or more amino acid substitutions.
 9. The method ofclaim 1, further comprising the step of engineering the conditionallyactive antibody to be multispecific.
 10. The method of claim 9, whereinthe selecting step selects the conditionally active antibody which alsoexhibits an affinity to an antigen in addition to an affinity for theblood-brain barrier receptor.
 11. The method of claim 1, wherein theblood-brain barrier receptor is selected from the group consisting of atransferrin receptor, an insulin receptor, an insulin-like growth factorreceptor, low density lipoprotein receptor-related protein 1, lowdensity lipoprotein receptor-related protein 8, and a heparin-bindingepidermal growth factor-like growth factor.
 12. The method of claim 1,wherein in the affinity for the blood-brain barrier receptor in theassay under the first physiological condition is measured by theconditionally active antibody's IC50 for inhibiting binding of theblood-brain barrier receptor's natural ligand, and the IC50 of theconditionally active antibody is from about 1 nM to about 100 μM. 13.The method of claim 1, wherein the evolving step comprises mutating a Fcregion of the antibody.
 14. The method of claim 1, wherein the evolvingstep comprises a technique selected from PCR, error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, gene site saturated mutagenesis, ligasechain reaction, in vitro mutagenesis, ligase chain reaction,oligonucleotide synthesis, and combinations thereof.
 15. The method ofclaim 1, wherein the expression step comprises expressing the mutant DNAin a host cell selected from a bacterial cell, a fungal cell, an insectcell, a mammalian cell, adenoviruses, and a plant cell.
 16. The methodof claim 15, wherein the host cell is a mammalian cell selected from aBowes melanoma cell, a COS-7 cell, a C127 cell, a 3T3 cell, a CHO cell,a HeLa cell and a BHK cell.
 17. A conditionally active antibody preparedby the method of claim 1, wherein the conditionally active antibody isreversibly inactivated under the second physiological condition.
 18. Theconditionally active antibody of claim 17, wherein the conditionallyactive antibody is conjugated to a molecule that is released under thesecond physiological condition.